3. are adherent cells (ie. cells in suspension are too far away from the coverslip)
|
|
- Ashley Woods
- 6 years ago
- Views:
Transcription
1 Before you begin, make sure your sample is seeded on #1.5 coverglass (thickness = 0.17) 2. is an aqueous solution (ie. fixed samples mounted on a slide will not work - not enough difference in refractive index of the glass and mounting solution for total internal reflection of the laser to occur) 3. are adherent cells (ie. cells in suspension are too far away from the coverslip) 4. has a fluorescent marker on the surface of the cell in order to focus on the sample. The adjacent to the coverslip will be used as a reference to focus on the coverslip (cannot focus or align the laser if there is no fluorescent markers at the coverslip interface) This training manual was adapted from the guide found at:
2 Total Internal Reflection Fluorescence Microscopy Total Internal Reflection Fluorescence Microscopy (TIRF) is... TIRF microscopy is a specialized fluorescence technique that images a very thin optical section (50-250nm) adjacent to the coverslip. TIRF microscopy is used to study molecular events at or near the cell surface with speeds and resolution that is not possible with other imaging techniques by using conditions that create total internal reflection that generates an evanescent wave. Basic Concepts of Total Internal Reflection Refractive Index: A measure in the reduction of the speed of light inside the a medium (compared to the speed of light in a vacuum) Refraction of Light: Vacuum 1.00 Air Water 1.33 Glass the bending or change in direction of light as it travels from one medium into another with different refractive indexes refraction of light only occurs when the incident light meets the interface at an angle light will travel straight through with no change of direction when crossing perpendicular to the interface the degree of refraction increases as the angle of the incident light increases Refraction Angle refraction Interface Aqueous Solution (RI=1.33) Glass (RI=1.52) Incident light Normal incident angle Critical Angle 90 Total Internal Reflection Critical Angle: the angle of the incident light where the refraction angle is 90 Total Internal Reflection: occurs when the incident angle is greater than the critical angle Majority of the light is reflected
3 Basic Concepts of Total Internal Reflection Continued... Evanescent Wave: during total internal reflection, a small portion of the reflected light penetrates through the interface this creates a very thin electromagnetic field (<250nm) adjacent to the interface (evanescent wave) this wave has identical frequency to the incident light the evanescent wave propagates parallel to the interface the intensity decreases exponentially with increasing distance away from the interface Evanescent Wave Interface Aqueous Solution (RI=1.33) Glass (RI=1.52) Critical Angle Total Internal Reflection decreasing intensity } <250nm This evanescent wave is used for excitation in TIRF microscopy
4 3. Total Internal Reflection Fluorescence Microscopy For TIRF microscopy... The laser is angled within the objective. Once the critical angle is passed, total internal reflection of the laser occurs and an evanescent wave is generated. The evanescent wave travels along the coverslip exciting the entire sample simultaneously (therefore the image can be acquired with a CCD camera) The intensity of the evanescent wave decreases exponentially away from the coverslip The evanescent wave only has sufficient energy for excitation within close proximity of the coverslip The evanescent wave only has sufficient energy for excitation within close proximity of the coverslip Therefore only fluorophores within this close proximity of the coverslip produce emission Fluorophores Evanescent Wave Fluorophores Excited Fluorophores Coverslip Coverslip CRITICAL ANGLE Objective Objective Laser Laser TIRF Sensor TIRF Sensor In the Leica system the returning beam of the total internal reflection is measured on an internal sensor. This feedback allows precise, fully automatic and reproducible setting of the penetration depth of the generated evanescent field
5 3. Total Internal Reflection Fluorescence Microscopy continued... TIRF microscopy is a tool to study the molecular events at or near the cell surface at a speed and resolution that is not possible with other imaging techniques. More specifically, TIRF microscopy is used to image at or within close proximity... Advantages of TIRF: distribution colocalization trafficking (movement in the membrane, endocytosis, exocytosis, etc.) changes in the above in response to various stimuli Fast image acquisition (up to 30 frames/sec) - the image is capture using a CCD camera since the evanescent wave propagates along the coverslip exciting the entire sample simultaneously Very thin optical section (50-250nm) adjacent to the coverslip Decrease signal-to-noise (increase contrast) Resolution MAY be improved with TIRF microscopy compared to confocal microscopy Resolution: the minimum distance between two points required to identify them as separate points The resolution limit of a microscopy is determined by: wavelength of light used for excitation numerical aperture (NA) of the objective R=0.61 /NA Example: R = (0.61x488nm)/1.4 = ~200nm 700nm 700nm z 200nm 250nm membrane(~10nm) 200nm 250nm coverslip x Confocal TIRF Confocal TIRF y 200nm 200nm y 200nm 200nm x Resolve as 2 separate structures No difference in resolution Resolve as 2 separate structures x Unable to resolve as 2 separate structures Improved resolution with TIRF due to thinner optical section Resolve as 2 separate structures
6 Comparison of TIRF microscopy with Epi-fluorescence and Confocal Microscopy TIRF EPI Confocal Evanescent Wave Excited Fluorophores Fluorophores Coverslip Focal Plane { } Focal Plane Laser TIRF Sensor Light Laser evanescent wave travels along the coverslip exciting the entire sample simultaneously fast image acquisition with CCD camera (30 frames/sec) No out-of-focus emission generated very thin optical section (50-250nm) decrease in signal-to-noise improving the contrast May improve resolution at the cell surface compared to confocal entire sample exposed to excitation light simultaneously fast image acquisition with CCD camera Both in-focus and out-of-focus emission collected No optical sectioning ( fuzzy image) High signal-to-noise (poor contrast) laser passes over the sample point by point slow image acquisition (25-30sec/image) Out-of-focus emission blocked by pinhole optical section ( nm) decrease in signal-to-noise improving the contrast focused on coverslip focused on coverslip focused at middle of cell (not the same cell as EPI & TIRF image)
7 A. THE EQUIPMENT The Leica DMI 6000B Inverted Microscope... Left Side Adjusts the intensity of the transmitted light and fluorescent light Controls the size of aperture diaphragm (leave fully open) Alters size of field diaphragm. (leave fully open) Toggles between Transmitted Light (TL) and Incident Light (IL, fluorescence) Front Light goes to camera Light goes to eyepiece Right Side GFP cube RFP cube QAD cube Move objective to lowest position
8 A. THE EQUIPMENT Continued... The Leica DMI 6000B Inverted Microscope adapted for TIRF microscopy... TIRF module - contains the scanner and TIRF sensor Incubation and laser safety box Laser box El6000 Fluorescence light supply Controls z movement of the objective Controls x,y movement of the stage SmartMove Precise or fast stage movement(x,y) Fine or Course focus (z movement)
9 B. START-UP PROCEDURE 1. Set the chamber temperature to 37 C Turn on Temperature Controller (Bottom Shelf) Turn on Heating Unit (On table, left side) IMPORTANT - The temperature controller MUST ALWAYS be on when the heating unit is on!!! The function of the heating unit is to heat and will continue to heat damaging the system if it is not regulated by the temperature controller It takes ~2hrs for the temperature of the entire system to equilibrate at 37 C. Make sure all of the doors and the top of the chamber is closed. 2. Switch on the system in the following order: A. Turn on El6000 Fluorescence supply (bottom shelf - right) B. Turn on the main power switch on the Laser box (green button on the black box left of the table) C. Turn on the camera (bottom shelf - left) D. Start the computer (and login) E. Move the condenser arm back and then turn on the microscope at the MICBOX (bottom shelf - middle). The stage will move and it is important to make sure the condenser will not be hit by the stage. F. Turn on the laser key switch (black box left of the table) G. Start the Leica LAS software Click on OK H. Intialize the stage? Yes if you want to use the Mosaic or Marking features, No if you do not. The stage will move if you select yes so make sure the objective and condenser will not be hit
10 There are 3 portions within the LAS Software: 1. Scan Parameters Window 2. Configuration Window 3. Image Window
11 C. SETTING THE CONFIGURATIONS OF THE MICROSCOPE In the Acquire tab and the Setup window: 1. Select the objective: HCX Plan-Apo 63x, NA 1.47 HCX Plan-Apo 100x NA 1.47 NOTE: the 40x is not a TIRF objective 2. In the Experiment Settings activate: Use sequencer board Use sequence timestamps Single image mode Enable Smartmove control during acquisition Z movement is irrelevant for TIRF 3. Select the type of Shutter Control: After each sequence for the fastest image acquisition 4. Set TIRF Configuration to: Automatic mode
12 D. FOCUSING ON THE SAMPLE The laser must be aligned at the start of every session. Before the laser can be aligned, there must be a sample in place and focused on the coverslip. This will be done using epi-fluorescence (FLUO). 1. Move the objective to its lowest position 2. Place a SMALL drop of oil on the objection (too much oil ruins objectives and the TIRF objectives are very expensive!!!!) 3. Place the sample in place and move the objective up with the focus knob until the oil makes contact with the bottom of the dish. 4. Close the doors on the chamber and let the temperature of the dish equilibrate (~10 min) 5. In the Light Path Settings, set the Contrast Method to FLUO GFP RFP QAD 6. Select the appropriate filter cube (GFP, RFP, or QAD) 7. Click on Live to open (and close) the shutter 8. Send the light path to the eye pieces 9. Select Course Focus on the SmartMove and focus the sample 10. Once in focus, send the light path back to the camera and switch back to Fine Focus on the Smartmove
13 D. FOCUSING ON THE SAMPLE Continued In the Acquisition Tab, adjust the settings of the camera - Binning - Exposure - Gain - EM Gain - Intensity (Set to 5 for TIRF) 12. Focus on the coverslip. NOTE: In order to align the laser properly (essential for good TIRF), you must focus on the coverslip. It is often very difficult to determine whether or not you are focused on the coverslip. As a suggestion, try to focus on the edges of the cells. 13. Click on Live to close the shutter
14 E. AUTOALIGNMENT OF THE LASER 1. Switch the Contrast Method to TIRF and select the GFP filter cube NOTE: All laser lines will be aligned regardless of the filter cube selected 2. Select Autoalign 3. Follow the instructions in the Autoalignment
15 F. ACQUIRING A TIRF IMAGE With the Contrast Method set for TIRF Select appropriate filter cube (QAD for rapid switching with multi-colour fluorescence) 2. Select the appropriate laser and adjust the laser power (start at 20%) 3. Select the appropriate pseudocolour 4 Select the penetration depth 5. To add another channel, click on + and select the appropriate filter cube, laser, pseudocolour and penetration depth (repeat step #1-4 for the new channel) NOTE: select the QAD filter cube for multi-colour fluorescence 5. Click on live to preview the image (click on Live again to turn off preview). GFP RFP QAD
16 G. OPTIMIZING THE TIRF IMAGE For each channel, adjust the following to optimize the image - binning - exposure - gain - penetration depth - direction of laser NOTE: In order for high speed image acquisition of multi-colours the filter cube, gain and direction of the laser must be same for all channels. Start and Stop preview for the active channel (and open shutter in Fluo) Capture a single image for the ACTIVE channel Capture a single image for ALL of the channels Start a time series
17 H. CAPTURING A TIME SERIES Once the settings have been optimized, 1. Click on t in the Acquisition Mode 2. Set the criteria for the time series (the interval between images and the duration) For high speed acquisition, select minimize and synchronize hardware NOTE: Synchronize hardware is only available if the filter cubes, gain and direction of the laser are identical for all of the channels. Start a time series
18 I. SHUT-DOWN PROCEDURE 1. Close LAS software and shut-down the computer 2. Turn the laser key off, but DO NOT turn the main power switch on the Laser box (green button) off yet. The laser must cool at least 5 min. 3. Switch off all of the other components in the system: - El6000 Fluorescence supply (bottom shelf - right) - Camera (bottom shelf - left) - Temperature Controller (bottom shelf) - Heating Unit (on table, left side) 4. Once the laser has cooled for at least 5 min switch of the main power switch on the laser box 5. CLEAN THE OBJECTIVE!!!! These objectives are very expensive and are damaged very easily if not cleaned properly. To clean: wipe off any oil on the objective with lens paper use lens paper damp with lens cleaner to clean the objective dry the objective with a clean and dry piece of lens paper IF YOU DO NOT CLEAN THE OBJECTIVES PROPERLY, YOUR MICROSCOPE PRIVILEGES WILL BE TAKEN AWAY. 6. Clean up any mess on the stage, in the incubator box, on the table and the desk.
Leica SP8 TCS Users Manual
Version : 07/08/0 Leica SP8 TCS Users Manual Start up:. Turn the PC Microscope, Scanner Power, Laser Power, and the Laser Emission key to on (bottom right of desk).. Turn on the fluorescent lamp (top left
More informationNikon Eclipse Ti2-E Widefield/Spinning Disk Confocal Microscope Standard Operation Protocol
Nikon Eclipse Ti-E Widefield/Spinning Disk Confocal Microscope Standard Operation Protocol Please sign on the log sheet before switching on system. Turn on system Turn on A only if confocal mode or laser
More informationLeica SP8 Resonant Confocal. Quick-Start Guide
Leica SP8 Resonant Confocal Quick-Start Guide Contents Start-up Preparing for Imaging Part 1 On the scope Part 2 Software interface Part 3 Heat & CO2 incubation Part 4 Other hardware options Shut-down
More informationGuide to Confocal 5. Starting session
Guide to Confocal 5 Remember that when booking and before starting session you can check for any problems at https://www.bris.ac.uk/biochemistry/uobonly/cif/index.html Starting session Switch on microscope
More informationTraining Guide for Leica SP8 Confocal/Multiphoton Microscope
Training Guide for Leica SP8 Confocal/Multiphoton Microscope LAS AF v3.3 Optical Imaging & Vital Microscopy Core Baylor College of Medicine (2017) Power ON Routine 1 2 Turn ON power switch for epifluorescence
More informationLeica TCS SP8 Quick Start Guide
Leica TCS SP8 Quick Start Guide Leica TCS SP8 System Overview Start-Up Procedure 1. Turn on the CTR Control Box, EL6000 fluorescent light source for the microscope stand. 2. Turn on the Scanner Power
More informationMotorized Axio Observer Start-up instructions
Start-up instructions 1. If using fluorescence turn on Fluorescent light source. TL light Source (Hal 100) 2. Turn on microscope using switch on lower left side of the microscope. 3. If imaging, turn on
More informationTRAINING MANUAL. Multiphoton Microscopy LSM 510 META-NLO
TRAINING MANUAL Multiphoton Microscopy LSM 510 META-NLO September 2010 Multiphoton Microscopy Training Manual Multiphoton microscopy is only available on the LSM 510 META-NLO system. This system is equipped
More informationLSM 710 Confocal Microscope Standard Operation Protocol
LSM 710 Confocal Microscope Standard Operation Protocol Basic Operation Turning on the system 1. Switch on Main power switch 2. Switch on System / PC power button 3. Switch on Components power button 4.
More informationQuick Start Guide. Leica SP5 X
Quick Start Guide Leica SP5 X Please note: Some of the information in this guide was taken from Leica Microsystems Leica TCS SP5 LAS AF Guide for New Users. This work is licensed under the Creative Commons
More informationLEICA TCS SP5 AOBS TANDEM USER MANUAL
LEICA TCS SP5 AOBS TANDEM USER MANUAL STARTING THE SYSTEM...2 THE LAS AF SOFTWARE...3 THE «ACQUIRE» MENU...5 CHOOSE AND CREATE A SETTING...6 THE CONTROL PANEL...8 THE DMI6000B MICROSCOPE...10 ACQUIRE ONE
More informationZeiss LSM 880 Protocol
Zeiss LSM 880 Protocol 1) System Startup Please note put sign-up policy. You must inform the facility at least 24 hours beforehand if you can t come; otherwise, you will receive a charge for unused time.
More informationOperating Checklist for using the Laser Scanning Confocal Microscope. Leica TCS SP5.
Smith College August 2010 Operating Checklist for using the Laser Scanning Confocal Microscope Leica TCS SP5. CONTENT, page no. Startup, 1 Initial set-up, 1 Software, 2 Microscope Specimen observation
More informationLSM 780 Confocal Microscope Standard Operation Protocol
LSM 780 Confocal Microscope Standard Operation Protocol Basic Operation Turning on the system 1. Sign on log sheet according to Actual start time 2. Check Compressed Air supply for the air table 3. Switch
More informationWidefield 1. Switching on
Widefield 1 Switching on 1. Ignite DG5 lamp - must be switched on first (if previous user has switched off, wait 30 min before igniting) 2. Wait 5s and then turn on the main DG5 controller switch. 3. DG5
More informationDiskovery Spinning Disk Guide
Diskovery Spinning Disk Guide qbi.microscopy@uq.edu.au Getting started The microscope and its peripherals (Fig. 1a) should always be turned on, but if they are not, turn them on in the following way: 1.
More informationWorking Simultaneously. The Next Level of TIRF Microscopy. cell^tirf Illuminator Motorized Total Internal Reflection Fluorescence
cell^tirf Illuminator Motorized Total Internal Reflection Fluorescence Four individually aligned illumination beams for simultaneous multi-color TIRF imaging Working Simultaneously The Next Level of TIRF
More informationTRAINING MANUAL. Olympus FV1000
TRAINING MANUAL Olympus FV1000 September 2014 TABLE OF CONTENTS A. Start-Up Procedure... 1 B. Visual Observation under the Microscope... 1 C. Image Acquisition... 4 A brief Overview of the Settings...
More informationLeica SP8 TCS Users Manual
Leica SP8 TCS Users Manual Follow the procedure for start up and log on as posted in the lab. Please log on with your account only and do not share your password with anyone. We track and confirm usage
More informationPractical work no. 3: Confocal Live Cell Microscopy
Practical work no. 3: Confocal Live Cell Microscopy Course Instructor: Mikko Liljeström (MIU) 1 Background Confocal microscopy: The main idea behind confocality is that it suppresses the signal outside
More informationSTART-UP PROCEDURE 1 THE MICROSCOPE STAND 3 OBJECTIVES 5 STARTING WITH LAS (SOFTWARE) AND SETTING UP THE MICROSCOPE STAND 7
Leica DMI AF6000LX Table of contents START-UP PROCEDURE 1 THE MICROSCOPE STAND 3 OBJECTIVES 5 STARTING WITH LAS (SOFTWARE) AND SETTING UP THE MICROSCOPE STAND 7 ACQUIRE MODULE 6 SETTING THE LIGHTPATH 6
More informationLeica TCS SP8 Quick Start Guide
Leica TCS SP8 Quick Start Guide Leica TCS SP8 System Overview Start-Up Procedure 1. Turn on the CTR Control Box, Fluorescent Light for the microscope stand. 2. Turn on the Scanner Power (1) on the front
More informationNikon. King s College London. Imaging Centre. N-SIM guide NIKON IMAGING KING S COLLEGE LONDON
N-SIM guide NIKON IMAGING CENTRE @ KING S COLLEGE LONDON Starting-up / Shut-down The NSIM hardware is calibrated after system warm-up occurs. It is recommended that you turn-on the system for at least
More informationOperation Guide for the Leica SP2 Confocal Microscope Bio-Imaging Facility Hunter College October 2009
Operation Guide for the Leica SP2 Confocal Microscope Bio-Imaging Facility Hunter College October 2009 Introduction of Fluoresence Confocal Microscopy The first confocal microscope was invented by Princeton
More informationZeiss LSM 780 Protocol
Zeiss LSM 780 Protocol 1) System Startup F Please note the sign-up policy. You must inform the facility at least 24 hours beforehand if you can t come; otherwise, you will receive a charge for unused time.
More informationThe Next Level of TIRF Microscopy. cell^tirf Illuminator Motorized Total Internal Reflection Fluorescence
cell^tirf Illuminator Motorized Total Internal Reflection Fluorescence Four individually aligned illumination beams for simultaneous multi-color TIRF imaging The Next Level of TIRF Microscopy Mario Faretta,
More informationThings to check before start-up.
Byeong Cha Page 1 11/24/2009 Manual for Leica SP2 Confocal Microscope Enter you name, the date, the time, and the account number in the user log book. Things to check before start-up. Make sure that your
More informationOverview. About other software. Administrator password. 58. UltraVIEW VoX Getting Started Guide
Operation 58. UltraVIEW VoX Getting Started Guide Overview This chapter outlines the basic methods used to operate the UltraVIEW VoX system. About other software Volocity places great demands on the computer
More informationTitle: Leica SP5 Confocal User Manual
Title: Leica SP5 Confocal User Manual Date of first issue: 23/10/2015 Date of review: Version: Admin For assistance or to report an issue Office: CG07 or 05 Email: Igmm-imaginghelpdesk@igmm.ed.ac.uk Website:
More informationOlympus Fluoview 1000S Spectral Confocal Microscope Introduction to the NRI-MCDB Microscopy Facility Spectral Confocal Microscope
Olympus Fluoview 1000S Spectral Confocal Microscope Introduction to the NRI-MCDB Microscopy Facility Spectral Confocal Microscope Improved Optics More Lasers 405 diode 440 diode 488 Argon 515 Argon 559
More informationRenishaw InVia Raman microscope
Laser Spectroscopy Labs Renishaw InVia Raman microscope Operation instructions 1. Turn On the power switch, system power switch is located towards the back of the system on the right hand side. Wait ~10
More informationLeica DMI 4000 tutorial
Leica DMI 4000 tutorial Before using the Leica DMI 4000, You will need to put down your name on the reservation system = 1 Welcome to the Leica DMI4000 Microscope tutorial How to start up the system (p.3)
More informationTraining Guide for Carl Zeiss LSM 5 LIVE Confocal Microscope
Training Guide for Carl Zeiss LSM 5 LIVE Confocal Microscope AIM 4.2 Optical Imaging & Vital Microscopy Core Baylor College of Medicine (2017) Power ON Routine 1 2 Verify that main power switches on the
More informationLeica Sp5 II Confocal User Guide
Leica Sp5 II Confocal User Guide Turning on the Confocal System (instructions are posted in the room) 1. Turn on Laser Power Button 2. Turn Key to On position 3. Turn on Scanner Power Button 4. Turn on
More informationUsing the Nikon TE2000 Inverted Microscope
Wellcome Trust Centre for Human Genetics Molecular Cytogenetics and Microscopy Core Using the Nikon TE2000 Inverted Microscope Fluorescence image acquisition using Scanalytic s IPLab software and the B&W
More informationNikon Ti-E Microscope Manual. Rightmire Hall Ohio State University. Director: Tony Brown Rightmire
Nikon Ti-E Microscope Manual Rightmire Hall Ohio State University Director: Tony Brown Rightmire 060 292-1205 brown.2302@osu.edu Facility Manager: Paula Monsma Rightmire 062 293-0939 292-1367 monsma.1@osu.edu
More informationZeiss Axio Imager.A1 manual
Zeiss Axio Imager.A1 manual Power-up protocol 1. Mercury lamp 2. Power strip on shelf 3. Computer The Mercury lamp should always be first-on and last-off. This prevents any electrical surges caused by
More informationEPIFLUORESCENCE &/OR BRIGHTFIELD MICROSCOPY
EPIFLUORESCENCE &/OR BRIGHTFIELD MICROSCOPY TURN ON THE FOLLOWING EQUIPMENT The fluorescent light (if needed) The power strip for the microscope and accessories The CoolSNAP HQ camera on the right (Turn
More informationLSM 510 Training Notes
LSM 510 Training Notes Turning on the system Turn on the arc lamp, found on the bench top left of the microscope. This supplies light for epifluorescence for viewing your samples through the microscope.
More informationTraining Guide for Carl Zeiss LSM 7 MP Multiphoton Microscope
Training Guide for Carl Zeiss LSM 7 MP Multiphoton Microscope ZEN 2009 Optical Imaging & Vital Microscopy Core Baylor College of Medicine (2017) Power ON Routine 1 2 Turn Chameleon TiS laser key from Standby
More informationZeiss 880 Training Notes Zen 2.3
Zeiss 880 Training Notes Zen 2.3 1 Turn on the HXP 120V Lamp 2 Turn on Main Power Switch Turn on the Systems PC Switch Turn on the Components Switch. 3 4 5 Turn on the PC and log into your account. Start
More informationBX-61: Brightfield Instruction /Continue to scroll for Fluorescent Instuctions
BX-61: Brightfield Instruction /Continue to scroll for Fluorescent Instuctions Starting up: Schematic of Olympus BX-61. 1. Turn on Olympus microscope power box (left of microscope) with toggle switch on
More informationLSM 800 Confocal Microscope Standard Operation Protocol
LSM 800 Confocal Microscope Standard Operation Protocol Turning on the system 1. Switch on the Main switch (labeled 1 and 2 ) mounted on the wall. 2. Turn the Laser Key (labeled 3 ) 90 clockwise for power
More informationLSM 510 Meta Training Notes
LSM 510 Meta Training Notes Turning on the system Turn on X-Cite power supply. This supplies light for epifluorescence for viewing your samples through the microscope. Turn on the remote control switch.
More informationZeiss 780 Training Notes
Zeiss 780 Training Notes Turn on Main Switch, System PC and Components Switches 780 Start up sequence Do you need the argon laser (458, 488, 514 nm lines)? Yes Turn on the laser s main power switch and
More informationBoulevard du Temple Daguerrotype (Paris,1838) a busy street? Nyquist sampling for movement
Boulevard du Temple Daguerrotype (Paris,1838) a busy street? Nyquist sampling for movement CONFOCAL MICROSCOPY BioVis Uppsala, 2017 Jeremy Adler Matyas Molnar Dirk Pacholsky Widefield & Confocal Microscopy
More informationNikon SIM-E & A1-R System
Nikon SIM-E & A1-R System USER GUIDE LSU Health Sciences Center Shreveport Research Core Facility June 01 2017 Chaowei Shang 1 Table of Content 1. Start Up the System... Page 3 Hardware and microscope
More informationSHORT INSTRUCTIONS FOR OPERATING LSM1/2 (Zeiss LSM510) AT CIAN Version 1.4, September 2014
CIAN LSM1 or LSM2 short instructions, version 1.4, September 2014 page 1 of 6 SHORT INSTRUCTIONS FOR OPERATING LSM1/2 (Zeiss LSM510) AT CIAN Version 1.4, September 2014 Before starting To work with LSM1
More informationZEISS LSM510META confocal manual
ZEISS LSM510META confocal manual Switching on the system 1) Switch on the Remote Control button located on the table to the right of the microscope. This is the main switch for the whole system including
More informationInstructions for the Leica SP5 II laser scanning confocal microscope
Instructions for the Leica SP5 II laser scanning confocal microscope Content: Check-in and Start up Set up acquistion parameters Optimize acquistion parameters Acquire a z-stack Sequential scan Check out
More informationQuick Guide for Zeiss 710 Laser Scanning Confocal MGH Cancer Center
Quick Guide for Zeiss 710 Laser Scanning Confocal MGH Cancer Center For any questions or concerns, please contact: Linda Nieman lnieman@mgh.harvard.edu Office: (617) 643-9684 Cell: (512) 565-8076 Chenyue
More informationTraining Guide for Carl Zeiss LSM 510 META Confocal Microscope
Training Guide for Carl Zeiss LSM 510 META Confocal Microscope AIM 4.2 Optical Imaging & Vital Microscopy Core Baylor College of Medicine (2017) Power ON Routine 1 2 Turn ON Components and System/PC switches
More informationLast updated: May 2014 Y.DeGraaf
FLINDERS MICROSCOPY BIOMEDICAL SERVICES AVAILABLE MICROSCOPES AND SPECIFICATIONS & INFORMATION REGARDING TRAINING FOR NEW USERS Last updated: May 2014 Y.DeGraaf If you have new staff or students (Honours/Masters
More informationTraining Guide for Carl Zeiss LSM 880 with AiryScan FAST
Training Guide for Carl Zeiss LSM 880 with AiryScan FAST ZEN 2.3 Optical Imaging & Vital Microscopy Core Baylor College of Medicine (2018) Power ON Routine 1 2 Turn ON Main Switch from the remote control
More informationNikon C1si Spectral Laser Scanning Confocal Microscope. User Guide
Nikon C1si Spectral Laser Scanning Confocal Microscope User Guide Contents: C1Si Turn-On/ShutDown Procedures... 2 Overview... 4 Setup for epi-illumination to view through the eyepieces:... 5 Setup for
More informationSupplementary Information. Stochastic Optical Reconstruction Microscopy Imaging of Microtubule Arrays in Intact Arabidopsis thaliana Seedling Roots
Supplementary Information Stochastic Optical Reconstruction Microscopy Imaging of Microtubule Arrays in Intact Arabidopsis thaliana Seedling Roots Bin Dong 1,, Xiaochen Yang 2,, Shaobin Zhu 1, Diane C.
More informationInternal Medicine Imaging Core Emory University Department of Medicine
Internal Medicine Imaging Core Emory University Department of Medicine 1 OPERATION OF THE ZEISS LSM 510 META YOU MUST SIGN UP TO USE THE MICROSCOPE OR COMPUTER EVERY TIME NO EXCEPTIONS Before attempting
More informationLeica SPEII confocal microscope. Short Manual
Leica SPEII confocal microscope Short Manual Switching ON sequence: 1. Turn on the Workstation under the bench (top, far right). 2. Turn on the Supply Unit - Laser box (big green switch first and then
More informationBi/BE 227 Winter Assignment #3. Adding the third dimension: 3D Confocal Imaging
Bi/BE 227 Winter 2016 Assignment #3 Adding the third dimension: 3D Confocal Imaging Schedule: Jan 20: Assignment Jan 20-Feb 8: Work on assignment Feb 10: Student PowerPoint presentations. Goals for this
More informationZEISS LSM 710 NLO Multiphoton microscope Manual/Quick guide
ZEISS LSM 710 NLO Multiphoton microscope Manual/Quick guide Matyas Molnar, Biovis 2016 Starting the microscpe 1. Check the microscope if everything looks clean and normal. If not, report it in the logbook.
More informationZeiss AxioImager.Z2 Brightfield Protocol
Zeiss AxioImager.Z2 Brightfield Protocol 1) System Startup Please note put sign-up policy. You must inform the facility at least 24 hours beforehand if you can t come; otherwise, you will receive a charge
More informationSwept-Field User Guide
Swept-Field User Guide Note: for more details see the Prairie user manual at http://www.prairietechnologies.com/resources/software/prairieview.html Please report any problems to Julie Last (jalast@wisc.edu)
More informationUser manual for Olympus SD-OSR spinning disk confocal microscope
User manual for Olympus SD-OSR spinning disk confocal microscope Ved Prakash, PhD. Research imaging specialist Imaging & histology core University of Texas, Dallas ved.prakash@utdallas.edu Once you open
More information3D light microscopy techniques
3D light microscopy techniques The image of a point is a 3D feature In-focus image Out-of-focus image The image of a point is not a point Point Spread Function (PSF) 1D imaging 1 1 2! NA = 0.5! NA 2D imaging
More informationQuick Guide for Zeiss 710 Laser Scanning Confocal MGH Cancer Center
Quick Guide for Zeiss 710 Laser Scanning Confocal MGH Cancer Center For any questions or concerns, please contact: Linda Nieman lnieman@mgh.harvard.edu Office: (617) 643-9684 Cell: (512) 565-8076 Chenyue
More informationConfocal Application Notes Vol. 5 July 2010
Tile Scan Prepared by Myriam Gastard, PhD Application and Technical Support Group, Leica Microsystems, Inc. In this issue of our Confocal Application Notes, proper set up of the Tile function enables you
More informationTitle: Nikon A1R Confocal User Manual
Title: Nikon A1R Confocal User Manual Date of first issue: 23/10/2015 Date of review: Version: Admin For assistance or to report an issue Office: CG.07 or CG.05 Email: Igmm-imaginghelpdesk@igmm.ed.ac.uk
More informationConfocal imaging on the Leica TCS SP8. 1) Turn the system on. 2) Use TCS user account. 3) Start LAS X software:
Confocal imaging on the Leica TCS SP8 1) Turn the system on. 2) Use TCS user account. 3) Start LAS X software: 4) Do not touch the microscope while the software is initializing. Choose your options: Turn
More informationNasmyth Ultraview Vox User Protocol
Nasmyth Ultraview Vox User Protocol Switch on all wall sockets labelled Nasmyth, switch camera on (power supply located on table behind monitor), switch on laser switch in laser rack, switch computer on
More informationRENISHAW INVIA RAMAN SPECTROMETER
STANDARD OPERATING PROCEDURE: RENISHAW INVIA RAMAN SPECTROMETER Purpose of this Instrument: The Renishaw invia Raman Spectrometer is an instrument used to analyze the Raman scattered light from samples
More informationOpterra II Multipoint Scanning Confocal Microscope. Innovation with Integrity
Opterra II Multipoint Scanning Confocal Microscope Enabling 4D Live-Cell Fluorescence Imaging through Speed, Sensitivity, Viability and Simplicity Innovation with Integrity Fluorescence Microscopy The
More informationSETTING UP OF A TOTAL INTERNAL REFLECTION FLUORESCENT MICROSCOPE (TIRFM) SYSTEM: A DETAILED OVERVIEW
PK ISSN 0022-2941; CODEN JNSMAC Vol. 51, (2011) PP 31-45 SETTING UP OF A TOTAL INTERNAL REFLECTION FLUORESCENT MICROSCOPE (TIRFM) SYSTEM: A DETAILED OVERVIEW A. R. KHAN 1 *, S. AKHLAQ 1, M. N. B. ABID
More informationTraining Guide for Carl Zeiss AxioZoom V16 Stereo Microscope
Training Guide for Carl Zeiss AxioZoom V16 Stereo Microscope ZEN 2012 Optical Imaging & Vital Microscopy Core Baylor College of Medicine (2017) Power ON Routine 1 2 If you require fluorescence imaging,
More informationZeiss Axiovert 135 Fluorescence Microscope Quick Guide / Operations Manual (v. 1.0 February 09)
University of Chicago Integrated Light Microscopy Core Dr. Vytas Bindokas, Director http://digital.bsd.uchicago.edu By: Christine Labno, Assistant Director Room: AB-129 Phone: 4-9040 Zeiss Axiovert 135
More informationNikon Instruments Europe
Nikon Instruments Europe Recommendations for N-SIM sample preparation and image reconstruction Dear customer, We hope you find the following guidelines useful in order to get the best performance out of
More informationZEISS LSM 710 CONFOCAL MICROSCOPE USER MANUAL
ZEISS LSM 710 CONFOCAL MICROSCOPE USER MANUAL START THE SYSTEM... 2 START ZEN SOFTWARE... 3 SET THE TEMPERATURE AND THE CO2 CONTROLLERS... OBSERVATION AT OCULARS... 5 STATIF PRESENTATION... 6 ACQUIRE ONE
More informationLeica TCS SL Confocal Training. Neuroscience Imaging Core Staff. Core Director. Facility Manager
Leica TCS SL Confocal Training Neuroscience Imaging Facility The Ohio State University Rightmire Hall 614-292-1367 Staff Core Director Anthony Brown, Ph. D. 060 Rightmire Hall 614-292-1205 brown.2302@osu.edu
More informationFLUORESCENCE MICROSCOPY. Matyas Molnar and Dirk Pacholsky
FLUORESCENCE MICROSCOPY Matyas Molnar and Dirk Pacholsky 1 The human eye perceives app. 400-700 nm; best at around 500 nm (green) Has a general resolution down to150-300 μm (human hair: 40-250 μm) We need
More informationUser Guide to the IBIF Leica TCS SP8 MP Confocal Microscope
User Guide to the IBIF Leica TCS SP8 MP Confocal Microscope This version: 7.24.14. Introduction The IBIF confocal microscope is made available on a fee-for-use-hour basis to all users who have been trained.
More informationFinal Exam, 150 points PMB 185: Techniques in Light Microscopy
Final Exam, 150 points Name PMB 185: Techniques in Light Microscopy Point value is in parentheses at the end of each question. Note: GFP = green fluorescent protein ; CFP = cyan fluorescent protein ; YFP
More informationZeiss LSM 510 Confocor III Training Notes. Center for Cell Analysis & Modeling
Zeiss LSM 510 Confocor III Training Notes Center for Cell Analysis & Modeling Confocor 3 Start Up Go to System Module Turn on Main Switch, System/ PC, and Components Switches Do you need the arc lamp?
More informationVISUAL PHYSICS ONLINE DEPTH STUDY: ELECTRON MICROSCOPES
VISUAL PHYSICS ONLINE DEPTH STUDY: ELECTRON MICROSCOPES Shortly after the experimental confirmation of the wave properties of the electron, it was suggested that the electron could be used to examine objects
More informationWidefield-NikonEclipseTE200-ORCA Nikon Eclipse TE200 Inverted Microscope with Hamamatsu 1394 Orca-ER Cooled CCD Camera and Micromanager Software
Widefield-NikonEclipseTE200-ORCA Nikon Eclipse TE200 Inverted Microscope with Hamamatsu 1394 Orca-ER Cooled CCD Camera and Micromanager Software September 2007 Check website for most current User Guide
More informationAkinori Mitani and Geoff Weiner BGGN 266 Spring 2013 Non-linear optics final report. Introduction and Background
Akinori Mitani and Geoff Weiner BGGN 266 Spring 2013 Non-linear optics final report Introduction and Background Two-photon microscopy is a type of fluorescence microscopy using two-photon excitation. It
More informationLSM 510 META in Chang Gung University
Content LSM 510 META in Chang ung University LSM 510 META 路 理 The features and applications of LSM 510 META 01-09 Introduction of the hardware 10-12 Fluorescence observation in conventional microscope
More informationWhy and How? Daniel Gitler Dept. of Physiology Ben-Gurion University of the Negev. Microscopy course, Michmoret Dec 2005
Why and How? Daniel Gitler Dept. of Physiology Ben-Gurion University of the Negev Why use confocal microscopy? Principles of the laser scanning confocal microscope. Image resolution. Manipulating the
More informationBrief manual how to start and close the Leica sp2 Confocal. (TCS SP2 AOBS system mounted on a DM IRE2)
Brief manual how to start and close the Leica sp2 Confocal (TCS SP2 AOBS system mounted on a DM IRE2) A. Switching on hardware B. Acquiring and saving images C. Switching off the microscope D. Good working
More informationNikon AZ100. Laser Scanning Macro Confocal Microscope. Jordan Briscoe Adam Fries Kyle Marchuk Kaitlin Corbin. May 2017.
Nikon AZ100 Laser Scanning Macro Confocal Microscope Jordan Briscoe Adam Fries Kyle Marchuk Kaitlin Corbin May 2017 Contents 1 Introduction 2 2 Hardware - Startup 2 3 Software/Operation 4 3.1 Multidimensional
More informationImaging Introduction. September 24, 2010
Imaging Introduction September 24, 2010 What is a microscope? Merriam-Webster: an optical instrument consisting of a lens or combination of lenses for making enlarged images of minute objects; especially:
More informationHoriba Jobin-Yvon LabRam Raman Confocal Microscope (GERB 120)
Horiba Jobin-Yvon LabRam Raman Confocal Microscope (GERB 120) Please contact Dr. Amanda Henkes for training requests and assistance: 979-862-5959, amandahenkes@tamu.edu Hardware LN 2 FTIR FTIR camera 1
More information1 Co Localization and Working flow with the lsm700
1 Co Localization and Working flow with the lsm700 Samples -1 slide = mousse intestine, Dapi / Ki 67 with Cy3/ BrDU with alexa 488. -1 slide = mousse intestine, Dapi / Ki 67 with Cy3/ no BrDU (but with
More informationOlympus LEXT OLS 4000 Confocal Laser Microscope
Olympus LEXT OLS 4000 Confocal Laser Microscope The Olympus LEXT OLS4000 is a confocal microscope capable of taking high-resolution 3D images. The magnification (Optical and Digital) of this microscope
More informationPractical Flatness Tech Note
Practical Flatness Tech Note Understanding Laser Dichroic Performance BrightLine laser dichroic beamsplitters set a new standard for super-resolution microscopy with λ/10 flatness per inch, P-V. We ll
More information3D light microscopy techniques
3D light microscopy techniques The image of a point is a 3D feature In-focus image Out-of-focus image The image of a point is not a point Point Spread Function (PSF) 1D imaging 2D imaging 3D imaging Resolution
More informationInstructions for Making On-Line Reservations for Microscopes in NB11-204
Instructions for Making On-Line Reservations for Microscopes in NB11-204 1. Log into Mail using Mail.swmed.edu 2. Log in using your university id and password. 3. Click the Calendar Tab at the top right
More informationMicroscopy from Carl Zeiss
Microscopy from Carl Zeiss Contents Page Contents... 1 Introduction... 1 Starting the System... 2 Introduction to ZEN Efficient Navigation... 5 Setting up the microscope... 10 Configuring the beam path
More informationThe Zeiss AiryScan System, Confocal Four.
The Zeiss AiryScan System, Confocal Four. Overview. The Zeiss AiryScan module is a segmented, radially stacked GaASP detector and collector system designed to subsample the airy disk of a point emission
More informationImaging Retreat - UMASS Customized real-time confocal and 2-photon imaging
Imaging Retreat - UMASS 2012 Customized real-time confocal and 2-photon imaging Mike Sanderson Department of Microbiology and Physiological Systems University of Massachusetts Medical School Thanks for
More informationUse of the HSW5 Spinning Disk Confocal Microscope Updated last May 25, 2010 OK
Use of the HSW5 Spinning Disk Confocal Microscope Updated last May 25, 2010 OK Getting Started: 2 Starting Micromanager and Loading a Configuration 3 The Main Micromanager GUI 3 Configuration Settings
More informationNature Protocols: doi: /nprot Supplementary Figure 1. Schematic diagram of Kőhler illumination.
Supplementary Figure 1 Schematic diagram of Kőhler illumination. The green beam path represents the excitation path and the red represents the emission path. Supplementary Figure 2 Microscope base components
More information