Last updated: May 2014 Y.DeGraaf

Transcription

1 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 or PhD) who require training in the use of the confocal, fluorescence and/or brightfield microscopes in Flinders Microscopy, please be informed trainings are held on Mondays and Tuesdays. Upon completed training, users are given access to the facility and the respective instruments they have received training on. All equipment use must be booked, and equipment charges known as user fees apply. User fees are kept to a minimum, and funds from user fees go back into the facility to cover repairs, part replacement and equipment upgrades. Confocal, brightfield and fluorescence microscope user fee charges are invoiced to supervisors bi-annually. Please refer to current table of user fees. Information on Flinders Microscopy can be found at and also Information on the various service sections within Flinders Microscopy are detailed below: For Electron Microscope (transmission, TEM) queries please contact Yvette.Degraaf@flinders.edu.au For Electron Microscope (scanning, SEM) queries please contact Daniel.Tune@flinders.edu.au For Atomic Force, Raman and Scanning Tunnelling Microscopy please contact Christopher.Gibson@flinders.edu.au or Joe.Shapter@flinders.edu.au For Immunohistochemistry Services queries please contact Yvette.Degraaf@flinders.edu.au or Pat.Vilimas@flinders.edu.au For Brightfield & Fluorescence Microscope queries Yvette.Degraaf@flinders.edu.au or Pat.Vilimas@flinders.edu.au For Confocal Microscope queries Yvette.Degraaf@flinders.edu.au

2 CONSIDERATIONS BEFORE REQUESTING TRAINING Please consider the following steps in brightfield, fluorescence and confocal protocols before you contact us. By considering all these aspects you will develop a better idea of how to construct your experiment and the required instrument. We can help you with this. You can print this page and write at each item on the cycle. Project Title: Sample GMO? Desired Image Outcome Targets/ Proteins/Antibodies Instrument Fluorophores or Stains or Labels Sample Proteins / Antibodies Fluorophores / Stains Instrument Desired Image Outcome tissue section user defined eg. TrkA receptor, actin filaments (phalloidin) DAPI / Hoescht Brightfield (Light) representative display only simple counting cells GFP / Alexa488 / FITC Widefield (Fluorescence) simple counting wholemount Cy3 / dsred / Alexa 594 Confocal (Fluorescence) percentage counting live cells Cy5 / mcherry / Alexa 647 qualitative colocalisation H&E / Toluidine Blue / peroxidase quantitative pixel intensity info quantitative colocalisation z-stack / movies / 3D projections

3 REQUESTING TRAINING When requesting training, please let Yvette know which instrument you are seeking training on Refer to the information on different instruments below and consider i) your specimen setup - mounted on a slide with a coverglass - in a tissue culture plate or flask - other (please discuss in advance of training) ii) whether or not your specimen is fluorescently labelled Please note that the brightfield, fluorescence and confocal microscopy section of Flinders Microscopy is not licenced for work with genetically modified organisms (GMOs) or infectious material. GMOs which are approved by the Flinders University Institutional Biosafety Committee as Excempt Dealings or NLRD PC1 may be bought into the facility following approval by Facility Management please request the appropriate form. Material requiring PC2 containment cannot be bought into the facility. If unsure please check with your supervisor. WHAT TO BRING TO A TRAINING SESSION Attendees should bring a suitably prepared specimen (see below) with them to the training session, with appropriate specimen transport. know what the specimen is, what target molecule has been labelled within the specimen, what the fluorescence (if applicable) label is, and what they are interested in investigating (see cycle chart above to help with this) NOT bring any genetically modified organisms, infectious material or chemicals with them without prior approval. TRAINING ON THE FLUORESCENCE AND BRIGHTFIELD MICROSCOPES is a single session and typically takes up to 1.5 hours includes an induction to the facility, OH&S, the booking system as well as use of the respective equipment and digital image acquisition software. TRAINING ON THE CONFOCAL MICROSCOPES takes significantly longer and usually spans 2-3 sessions of 2-2½ hours each (depending on the complexity of the work and competence/experience of the user) For short-term students/projects it may be more feasible and cost-effective for Flinders Microscopy staff to operate the confocal microscope for them rather than to be trained to use it themselves. DID YOU KNOW? FLINDERS MICROSCOPY BIOMEDICAL SERVICES NOW OFFERS IMMUNOHISTOCHEMISTRY SERVICES. We can provide advice and bench space and consumables for researchers to set up immunohistochemistry experiments and also offer fluorescence immunohistochemistry services provided by an experienced technician for a fee. Contact Pat Vilimas or Yvette DeGraaf for further information

4 OLYMPUS BX50 FLUORESCENCE MICROSCOPE (upright microscope) Most first-time users who require fluorescence microscopy are trained on this upright microscope unless there is a particular reason for using another microscope. This microscope is suitable for imaging most common blue, green, red and far-red dyes (refer to Filtercube Specifications document), and has 4x, 10x, 20x and 40x dry objective lenses (40x to 400x total magnification). Regular transmitted light imaging is also possible. Specimens for this microscope should be mounted on a glass slide with a coverslip, and sealed (eg with nail and dry, without excess mounting medium, and nail polish/hard set mounting media needs to have had time to completely set (1 hour for nail polish). AX70 FLUORESCENCE OLYMPUS MICROSCOPE (upright microscope) This microscope is suitable for mounted slides, and is suitable for imaging most common blue, green, red and far-red dyes (but has some minor differences in the specifications of the filter cubes compared to the Fluorescence BX50 microscope, refer to Filtercube Specifications document). This microscope has a 40x and 100x oil objective lens as well as 4x, 10x, 20x and 40x dry objective lenses (40x to 1000x total magnification). Regular transmitted light imaging and DIC (differential interference contrast) imaging is available. A live video camera is also available which may help in locating far-red fluorophore labelling, which is invisible to the human eye unless it is exceedingly bright. Specimens for this microscope should be mounted on a glass slide with a coverslip, and sealed (eg with nail and dry, without excess mounting medium, and nail polish/hard set mounting media needs to have had time to completely set (1 hour for nail polish). OLYMPUS IX71 FLUORESCENCE & CONSTRAST MICROSCOPE (inverted microscope) This microscope is suitable for fluorescence or brightfield/contrast imaging of live or fixed cells in tissue culture flasks/plates. It is suitable for imaging common blue, green, red and far-red dyes (see Filtercube Specifications document) as well as phase contrast and relief contrast imaging. It is equipped with 10, 20x and 40x dry objective lenses (100x to 400x total magnification). Cells should be correctly transported, flask/plate within an appropriate container, and, for GMOs, handled in accordance with IBC requirements. Note that PC2 work cannot be brought into Flinders Microscopy. If you are unsure about the suitability of bringing your specimen into the facility to image with this microscope please contact Facility Management. Please consult with Facility Management regarding the use of the cell culture incubator. BRIGHTFIELD OLYMPUS BX50 MICROSCOPE (upright microscope) This microscope is suitable for mounted slides where fluorescence is not required. It is equipped with a digital colour camera, and 2x, 4x, 10x, 20x and 40x dry objective lenses and a 100x oil objective lens. Multiple condensers are used on this microscope depending on the chosen lens; training therefore includes being shown how to change condensers and how to set the correct condenser height and field diaphragm aperture to achieve optimal illumination ( Koelher Illumination ). Specimens for this microscope should be mounted on a glass slide with a coverslip, and sealed (eg with nail and dry, without excess mounting medium, and nail polish/hard set mounting media needs to have had time to completely set (1 hour for nail polish). SZX10 STEREO MICROSCOPE (dissecting microscope with reflected light illumination) This microscope is suitable for low magnification (6.3x to 63x total magnification) imaging with reflected light, and may be used for a range of specimens. Specimens need to be transported appropriately, and, for GMOs, handled in accordance with IBC requirements. Note that PC2 work cannot be brought into Flinders Microscopy. If you are unsure about the suitability of bringing your specimen into the facility to image with this microscope please contact facility management.

5 CONFOCAL MICROSCOPES (Leica SP5 inverted, Olympus FV1000 inverted & BioRad MRC2400 upright). Confocal microscopes provide "optical slice" images (z-sections) of fluorescence, with out-of-focus light removed, providing improved resolution in the depth of the image (z-dimension). The capture of a series of such z-sections (z-stack) generates a 3D data set from a specimen. Achievable resolution and the depth within a specimen from which high resolution optical slice image can be obtained depends on the optics of the microscope, refractive indices through the sample and the depth of penetration of the labelling. Our confocal microscopes are all equipped with the highest quality lenses. With good sample preparation and minimum refractive index mismatch, resolution of up to 200 x 200nm in x & y and 500nm in z can be achieved. Resolution in z can be further improved with post image acquisition deconvolution (discuss prior to capturing images if you intend to do this). Imaging to a depth of up to ~50um can be achievable in specimens that present minimal refractive index mismatch and good label/antibody penetration, but ideally the part of the specimen to be imaged should be as close to the coverglass as possible, preferably mounted or grown on the coverslip, then sealed onto a slide. The Olympus FV1000 instrument is owned by Vaxine, who have generously agreed for it to also become available for common service use. It is housed within Flinders Microscopy 4E500 and became available for common service use in the second half of People seeking confocal training should have a clear idea of what they would like to achieve, and should first check that their fluorescence labelling has worked on a regular fluorescence microscope. Prospective confocal users should therefore have received prior trained on a fluorescence microscope unless they have one available in their own lab, or this is not suitable for their specimens. It is also possible to have Flinders Microscopy staff operate a confocal microscope on the users behalf, and in some situations such as one-off or short term microscope projects this may be more time and cost efficient than training a new user to operate it themselves. Please check with facility management regarding how to appropriately prepare live cells for confocal imaging, as it is best to receive prior advice on ways specimens can be set up to suit the respective microscope chambers and stages. Independent users who have fixed cell confocal experience need to consult with Facility Management before progressing to live cell imaging to ensure optimal setup and correct use of equipment for this application, objective lenses and microscope setup for live cell imaging requires brief additional training. For initial confocal microscope training, a fixed, mounted specimen is recommended, ie, specimens should be mounted on a glass slide with a coverslip, and sealed (eg with nail and dry, without excess mounting medium, and nail polish/hard set mounting media must have had time to completely set (1 hour for nail polish). Any texta markings to locate regions of interest within a specimen should be annotated on the slide, not the coverglass. Our confocal microscopes are suitable for fluorescence imaging of any fluorophore requiring excitation between near UV and red (choice of excitation between 405nm to 633nm) and any emission between 400 and 800nm. All three confocal microscopes feature adjustable scan speed, pixel format and optical zoom, and have very high quality, high numerical aperture (and very expensive) objective lenses. The Leica SP5 and Olympus FV1000 can also be used for a variety of live cell imaging (in addition to fixed cell imaging), including ph and ion imaging, FRET, FRAP, FLIP, FCS and RICS. A similar Leica SP5 at Adelaide Microscopy can be used for FLIM. The Leica SP5 and Olympus FV1000 can also perform transmitted light imaging, with or without contrast optics. Details on the lasers and emission detection of the 3 confocal microscopes is provided below. The main points of difference are that the Leica SP5 has 5 fully tuneable detectors (can set desired emission range for a given fluorophore, rather than being locked into available filter cube emission bandwidths), additional laser lines for CFP & YFP imaging, and additional single photon sensitive filtercube-based detectors and resonant scanner for where extremely high sensitivity or high speed scanning is required. It is also suitable for short term live cell experiments. The microscope of the Olympus FV1000 is housed within in a fully enclosed gas and temperature controlled chamber, and is on a gas-floated anti-vibration table (superior to the anti-vibration table our Leica SP5 is on), which, in conjunction with its autofocus options and silicon-immersion lenses make it the confocal of choice for medium to long term live cell experiments. The superior stability of the gas-floated anti-vibration table is an advantage for experiments where stage stability is critical, eg for FRET-acceptor bleaching and FRAP measurements.

6 Objectives, laser lines and emission detection: The Leica SP5 has 8 laser lines; 405, 458, 476, 488, 496, 514, 561 & 633nm and 5 fully adjustable PMT detectors (ie can be set to capture any given range of emission from 400 to 800nm), transmitted light detector and 2 filter cube based avalanche photo diode (single photon sensitivity) detectors which can be used with green and far-red fluorophores for imaging or FCS. 10x & 20x multi-immersion fluid lenses, 40x, 63x & 100x oil immersion lenses and a 63x water immersion lens are available. The Olympus FV1000 has 4 laser lines; 405, 473, 599, & 635nm lasers, and 3 PMT detectors for emission capture through fixed filter cubes suitable for most blue, green, red and far red dyes, and an additional transmitted light detector. 10x, 20x & 40x dry lenses, 30x & 60x silicon-oil immersion lenses. The Biorad MRC 1024 has 3 laser lines; 488, 568 and 647nm, and 3 PMT detectors for emission capture through fixed filter cubes suitable for most green, red and far red dyes. 10x, 20x, 40x, 60x and 100x oil lenses are available, as well as 4x and 40x long working distance dry lenses Flinders Microscopy Biomedical Services Flinders University Yvette DeGraaf, Microscopy & Cell Biology Support, , yvette.degraaf@flinders.edu.au Pat Vilimas, Immunohistochemistry Support, , pat.vilimas@flinders.edu.au Physical location: Flinders Medical Centre, Level 4, Room 4E500 Website: