FLUORESCENCE MICROSCOPY

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1 FLUORESCENCE MICROSCOPY Methods for Cell Analysis Course BioVis Uppsala, 2015 Matyas Molnar and Dirk Pacholsky 1

2 Information This lecture contains images and information from the following internet homepages

3 3

4 Light phenomenon 4

5 Light phenomenon 5

6 The human eye perceives app nm; best at around 500 nm (green) Has a general resolution down to μm (human hair: μm) We need a tool to see smaller things or more of the spectral range Microscope (Objective/Filter) Camera/Film 6

$000 km/sec Seems to slows down in denser matter. Refractive index n = C/v (v= speed in matter) light is 1.5 x slower at n=1.$

7 Light phenomenon Light photon particle and electromagnetic wave. λ E White light comprises of light of different λ (nm,color). Light travels in vacuum C = km/sec Seems to slows down in denser matter. Refractive index n = C/v (v= speed in matter) light is 1.5 x slower at n=1.5 Light changes direction between different dense materials : shorter λ refract more than longer λ Optic lenses, effects in sample... shorter wavelength (blue) has higher energy 7

8 WHY FLUORESCENCE MICROSCOPY? 8

9 Microscopical techniques Brightfield - low contrast for thin or transparent specimen - staining to enhance contrast needed (histochemical staining) Phase contrast - contrast via optical element (Phase ring) - intracellular structures can be seen - good for cell culture applications - negative: halos around cell bodies - combining with other techniques is generally poor (e.g. overlay with fluorescent image) 9

Microscopical techniques DIC/Nomarski - contrast via polarized light & optical

combination with fluorescence and histochemical staining Fluorescence - contrast

..in the perfect world) - special optical elements are needed (filter cubes) high

10 Microscopical techniques DIC/Nomarski - contrast via polarized light & optical element (Wollaston prism) - gives a (fake) topographical view - excellent for combination with fluorescence and histochemical staining Fluorescence - contrast via fluorescent staining - You see only what you stain (...in the perfect world) - special optical elements are needed (filter cubes) high resolution, high contrast, good for quantification (area + intensity) - Staining is sensitive - it can fade. 10

11 THE MICROSCOPE 11

12 Optical pathway of a microscope Objective projects image of specimen via Tube lens to Primary image plane Eyepiece magnifies this image. * Objectives are not interchangeable 12

13 Optical pathway of a microscope 1) Illuminator light source and different filters 2) Light conditioner e.g. phase annulus, FL excitation filter, polarizer 3) Condensor e.g. resolution, aberration 4) Specimen properties of specimen, immersion media, CS 5) Objective e.g. resolution, magnification, aberration working distance 6) Image filter e.g. Phase plate, FL emission filter, analyzer 7) Eyepiece e.g. magnification, field size 8) Detector human eye, camera 13

14 Optical pathway of a microscope 14

The objective or lens The heart of a microscope, may

(resolution) - Immersion medium (should fit to embedding

15 The objective or lens The heart of a microscope, may contain up to e.g. 12 lenses Specification and Identification - Magnification (enlargement) - Numerical aperture (resolution) - Immersion medium (should fit to embedding medium) - corrections (spherical; chromatical) - working distance - tube length (infinity or 160 mm) - coverslip thickness 15

Objective magnification and resolution A

75 0.37 µm resolution 40x N.A. 0.75 0.37 µm resolution 60x N.

16 Objective magnification and resolution A microscope magnifies a specimen with a certain resolution. 10x / high NA 10x / low NA objectives 20x N.A µm resolution 40x N.A µm resolution 60x N.A µm resolution Magnification without resolution is useless : empty magnification! 16

17 Illumination of the specimen Lens Light to/ from point source focal point in XZ Sample Z view Focal point Focal plane resolution in XY approx 2 x better than in Z The better the objective the smaller the focal point better means Numerical Aperture See next slide Field of view 17

$light 400-700 nm) n: refractive index (air: 1, water: 1.3, oil: 1.4-1.$

18 Illumination of the specimen - resolution d = λ 2nsin θ d: point resolution, the shorter the lenght is better for us [nm] λ: wavelength of light used ([nm], visible light nm) n: refractive index (air: 1, water: 1.3, oil: ) ѳ: the maximum cone of light that can enter or exit the lens nsin(2 ѳ) : numerical aperture (NA) 18

19 Illumination of the specimen Lens hour glass shaped light cone indicates N.A. Larger N.A. Collects more photons sample Z direction Focal plane F Information (light) coming from above/below focus disturbs focus information. unsharp images get overlaid with sharp images from focus Blurred image in total Get rid of that extra to see only information from Focal plane by Calculations (e.g. Deconvolution) or Technique LSM...PINHOLE 19

Objective aberrations Chromatic aberration:

$travels with different speed (refraction)$ Spherical abberations: unsharp images Different

20 Objective aberrations Chromatic aberration: color fringes light in different wavelength travels with different speed (refraction) therefore they won t meet in one focal point Spherical abberations: unsharp images Different focus of paraxial and peripheral light rays Corrections are possible 20

21 Objective aberrations - correction 21

22 Objective names Achromat, Planachromat chromatical corrected for RB, spherical corrected RB Fluar, Fluorite, Planfluorite chromatical corrected for RGB, spherical corrected RB Apochromat, Planapochromat high N.A. objectives, chromatical corrected for RGB+UV, spherical corrected RGB Different brands will have their own (confusing and abbreviated) objective names Planapochromates are the objectives to use if resolution and high quality photomicroscopy is needed. Plan objectives: normal lenses project a curved image. Plan lenses are flat-field corrected, both the center and the edge of the field is in focus. Plan-Neofluar is a fine universal objective for FL-microscopy. Use correct coverslip (1.5 = 170µm thickness), immersion for best imaging. 22

23 Resolution, Airy disk, NA & WD WD 23

will not be a point again in the image, but rather a dot (1st maxima, AiryDisk) with several side maxima separated by mininima (interference

24 Objective Resolution and Airy disk A point of light will not be a point of light Light originally coming from a point and passing through lenses etc. will not be a point again in the image, but rather a dot (1st maxima, AiryDisk) with several side maxima separated by mininima (interference pattern). he yellow dots shall indicate infinite points, where light originally came from. z Airy disk in XZ x The Spreading from Point light source to a Airy disk Image is called Point Spread Function (PSF). PSF gets bigger with mismatch of embedding medium and objective

25 Objective NA - Resolution and Airy disk One gains better Optical resolution by Increase of Numerical aperture (N.A.) of the objective used 25

26 Wavelength- Resolution and Airy disk One gains better Optical resolution by Decrease of wavelength of fluorophore used maximum resolution is approx µm lateral and 0.40 µm axial 26

$Light coming from high to low density medium (glas vs air) gets refracted away from the vertical of the incident angle, eventually misses the lense and is lost for$ $Appliance of immersion (oil, glycerin, water) between Coverslip and Lense with similar refractive index as Glas will reduce refraction and enhance light yield which in$

27 Match embedding medium to objective. Light coming from high to low density medium (glas vs air) gets refracted away from the vertical of the incident angle, eventually misses the lense and is lost for imaging. Appliance of immersion (oil, glycerin, water) between Coverslip and Lense with similar refractive index as Glas will reduce refraction and enhance light yield which in turn gives better Airy pattern (resolution) Light coming from one point source will get scattered and refracted into different angles the point gets spreaded. By applying High Numerical Aperture this effects are kept to a minimum. PSF gets bigger with mismatch of embedding medium and objective

28 Practical tips Bad approach Match objective with correct - Immersion media - Coverslip thickness - Embedding media - Imaging setup - Sample preparation good approach - Objectives indicate for which immersion media they are made for - Objectives indicate for which coverslip thickness they are made for - Embedding medium has optimally same RI like immersion media - Place the sample as close as possible towards the objective lens - RI of sub-cellular components considerably lower than that of immersion media, and in many cases these RI are uncertain and vary throughout the specimen. Different fixations might destroy antigen to be targeted or might quench fluorescence (of e.g. GFP)

29 Resolution summary BEST RESOLUTION - NA as high as possible (1.4 max) - Refractive index of embedding medium like glass /oil - Use of immersion oil objective - Excitation/emission wavelength as short as possible (shift to blue region) d = λ 2nsin θ 29

30 Applications for fluorescent probes Proteins using antibodies Receptors using conjugated ligands DNA RNA Lipids Lectins to detect proteoglycans and glycolipids Cytoskeleton Organelles Tracers for cells and fluids Viability, proliferation Ions (Ca 2+,,Mg 2+, Zn 2+,Na +, K +, Cl -.) ROS ph Membrane potential 30

31 THE FLUORESCENCE MICROSCOPE 31

32 Fluorescence Examples of fluorescent probes Principle of fluorescence Principle of fluorescent microscope Excitation-Emission filter cube

band-pass-filter to choose wavelength for

33 The fluorescence microscope Human eye perceives nm Camera/detector will do better mercury lamp Mercury (Xenon) lamp spectrum: (1300) nm Filtercube with band-pass-filter to choose wavelength for Excitation and Emission, including a special (dichroic) mirror.

34 Fluorescent dye spectra Spectra always bell shaped Normalized Intensity Alexa 488 Excitation spectra Which wavelength is best suited to excite this fluorophore? At 490 nm 100 % of fluorophore will get excited, but only 20% at 450 nm Emission spectra Wavelength of output emission it stays the same independent whether excitation was done with 490 nm or 450 nm BUT emission intensity will be lowered. nm

35 Dealing with fluorescence Cell sample Cell images merged RGB image 35

36 Dealing with fluorescence ex Cell sample Cell image* Excitation 350 nm excitates Blue and Green, using BP filter collects them both. *Remember: the camera is color blind. You decide with your choice of filter what it will see. 36

37 Dealing with fluorescence ex Cell sample Cell image* Excitation 350 nm excitates Blue and Green, using BP filter collects only the blue. *Remember: the camera is color blind. You decide with your choice of filter what it will see. 37

38 Dealing with fluorescence ex Cell sample Cell image* Excitation 480 nm excitates Green and Red, using BP filter collects only the green. *Remember: the camera is color blind. You decide with your choice of filter what it will see. 38

Combining fluorescent dyes - crosscheck To avoid false positive images in Fluorescence microscopy check for Seeing is Believing BUT Is it true?

39 Combining fluorescent dyes - crosscheck To avoid false positive images in Fluorescence microscopy check for Seeing is Believing BUT Is it true? What s to be seen in pos/neg control stained unstained Crossreact AbX with AbY? AbX AbY(1) X Y AbX X Y AbY(2) Unspecific backgr. by Ab)? cell with - w/out target X X Appropr. fixation? Fixation A) X Fixation B) Crosstalk/ Bleeding through? ex Use quality objectives, correct filter, embedding medium 39

40 Widefield vs Optical section Kidney sample 10µm thick, 63x/NA 1.43, Widefield image and optical section using Apotome technique. 40

Bleaching Bleaching before and after 100x imaging same area with

Test sample is a strong stain and so bleaching might be subtle and

emission are shown in LUT Black to white LUT= Blue, over green,

41 Bleaching Bleaching before and after 100x imaging same area with Widefield microscopy. Test sample is a strong stain and so bleaching might be subtle and only clearly be see in LUT (look-up-tables) Intensities of emission are shown in LUT Black to white LUT= Blue, over green, yellow, red You might not see the subtle changes But would like to compare Intensities Between image 1 and 2? Be aware... 41

42 IMAGING 42

Imaging digital camera - pixel 0-255 (8bit) All pixels of the

pixels at once Black&White cameras pixel does not care about color.

43 Imaging digital camera - pixel (8bit) All pixels of the camera will be exposed to light at once; image is processed all pixels at once Black&White cameras pixel does not care about color. For Fluorescence microscopy use B&W cameras (with appropriate filtercubes) 43

44 Imaging Features of a digital camera Spatial Resolution: ability to capture fine specimen details without pixels being visible in image (1308x1040 pixel, 6.45x6.45µm pixel on 2/3 chip) Light-Intensity Resolution: dynamic range or number of gray levels that are distinguishable in image. (12 bit or 16 bit) Time Resolution: frame rate - the ability to follow movement or rapid kinetic processes (38 fps) Signal-to-Noise Ratio: visibility and clarity of specimen signals relative to the image background Spectral Sensitivity: range of wavelength on which camera reacts ( nm) (data from Zeiss Axiocam MRm) 44

45 Image quality over/underexposure Sample :3 color staining balanced imaging over/underexposure palette mode visualizing Over/under exposure Remember: all color is based on grey value Red: overexposed Blue: underexposed 45

Image quality dynamic range Acquire your image

46 Image quality dynamic range Acquire your image with appropriate grey level (8bit at least) to represent different intensity levels. Incoming photons 12 or 16 ( grey levels )bit images would also allow you e.g. to enhance features lying in the dark to stretch them into the light... 46

47 Image quality - resolution example for aliasing and pixel blocking Image should not appear pixelated have enough pixels not enough pixels result in aliasing (jagged edges) and pixel blocking More pixels needed? Larger Chip more expensive camera Smaller pixels size less sensitive, signal to noise ratio decreases Scientific cameras 6.45x6.45 µm pixel LSM-Systems variable pixel size 47

Rest of pixels are calculated in respect to surrounding pixels (+/-). i.e. 66% (2 of 3 colors /px). More green in the Bayer mosaic, therefore human eye is more sensitive to green.

48 Imaging color vs black&white In Color Cameras each pixel is overlaid by color filter lense pattern The Bayer mosaic. Reduction of sensitivity and actual resolution + +/- +/- +/ Color pixel red only lets pass light in red range (+) signal. Rest of pixels are calculated in respect to surrounding pixels (+/-). i.e. 66% (2 of 3 colors /px). More green in the Bayer mosaic, therefore human eye is more sensitive to green. Problem: Actual resolution is 2x2 pixel i.e. 4x less Solution: camera with moveable chip are used each pixel will sample light from (9) different positions. High resolution Brightfield Black&White cameras pixel does not care about color. For Fluorescence microscopy use B&W cameras (with appropriate filtercubes) 48

Publishing photos Use the highest bit depth as possible, and take care about over/underexposure Use the highest quality/resolution as possible Use TIF files Crop image if neccessary Use measure bar

49 Publishing photos Use the highest bit depth as possible, and take care about over/underexposure Use the highest quality/resolution as possible Use TIF files Crop image if neccessary Use measure bar to show scale Do not use total magnification, e. g. objective magnification - 60X (which is of course not 60X magnification but 600X at least) Magnification 1000x VS Measure bar ( light years) 49

50 THANKS FOR YOUR ATTENTION! 50

FLUORESCENCE MICROSCOPY. Matyas Molnar and Dirk Pacholsky

FLUORESCENCE 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