Live Specimen Microscopy

Transcription

1 Jens Jens Rietdorf: Rietdorf: This This presentation presentation is is meant meant give give some some general general hints hints for for live live specimen specimen microscopy microscopy Live Specimen Microscopy

2 Contents Environment Physical integrity Attachment Temperature Gases (CO 2, O 2,H 2 O), ph. Osmolarity Illumination Autluorescence Phodamage Microscopy image processing techniques deconvolution Live specimen microscopes (Time lapse sequence analysis)

3 Jens Jens Rietdorf: Rietdorf: There There different different possibilities possibilities guarantee guarantee physical physical integrity integrity specimen specimen during during imaging. imaging. Physical integrity

4 Jens Jens Rietdorf: Rietdorf: Some Some tricks tricks in in case case sample sample does does not not adhere. adhere. Tissues Tissues or or embryos embryos can can be be trapped trapped under under transpnt, transpnt, gas gas permeable permeable plastic plastic films. films. Bacteria Bacteria or or nonadherent nonadherent cells cells can can be be embedded embedded in in or or overlayed overlayed with with 0.1% 0.1% low low meltingpoint meltingpoint (LMP) (LMP) agarose agarose Attachment Acid cleaning or flaming coverglass Withdraw serum Coating coverglass Poly-L-Lysine Concanavalin A Or 0.1% LMP agarose Transpnt films

5 Jens Jens Rietdorf: Rietdorf: Temperature Temperature is is critical critical both both for for integrity integrity sample sample stability stability microscope. microscope. Several Several solutions solutions discussed. discussed. Temperature

6 Jens Jens Rietdorf: Rietdorf: An An example example movie movie a a TIRF TIRF timelapse timelapse under under bad bad temperature temperature control. control. The The feedback feedback is is o o slow. slow. Temperature Temperature shifts shifts in in order order 0.1degC 0.1degC visible visible with with highna highna lenses. lenses. Example Focusshift

7 Gases (CO 2, O 2,H 2 O), ph, osmolarity Replace carbonate buffer inside medium by HEPES (e.g. 30mM HEPES, 0.5g/l Carbonate instead 2.2g/l Carbonate). Seal sample chamber (no gas exchange) Control CO 2, evaporation Use perfusion chambers Use incubars Jens Jens Rietdorf: Rietdorf: Regunly Regunly culture culture media media contain contain carbonate carbonate buffers buffers which which only only stable stable under under 5% 5% CO2 CO2 atmosphere. atmosphere. Examples Examples open open closed closed incubation incubation chambers discussed.

8 HEPES buffered media Advantages Open system, easy manipulate. Easy hle control. Disadvantages Usable for ca. 1 hour. Toxic conversion HEPES by irradiation. Evaporation.

9 Sealed chambers Advantages Easy hle control. No evaporation. Cheap. Disadvantages Usable for max 3 hours depending on volume. No manipulation.

10 Perfusion chambers Advantages Constant conditions. Manipulation media. Usable for days. Disadvantages Hard assemble control. Expensive.

11 Microscope Incubars Advantages Constant conditions. Manipulation. Usable for days. Expensive. Disadvantages Microscope access impaired.

12 Jens Jens Rietdorf: Rietdorf: Example Example movie movie good good environment environment control. control. 72hour 72hour spanning spanning timelapse timelapse without without focus focus shift, shift, cells cells divide divide express express gfp gfp which which is is good good indication indication y y in in good good shape. shape. Glasgow

13 Contents Environment Physical integrity Attachment Temperature Gases (CO 2, O 2,H 2 O), ph. Osmolarity Illumination Autluorescence Phodamage Microscopy image processing techniques deconvolution Live cell microscopes (Time lapse sequence analysis)

14 Jens Jens Rietdorf: Rietdorf: Autluorescence Autluorescence may may have have different different reasons, reasons, but but is is generally generally stronger, stronger, shorter shorter wavelength wavelength higher higher intensity intensity excitation excitation light light is. is. Stressed Stressed or or damaged damaged specimen specimen show show AF. AF. Autluorescence Specific sources autluorescence (excitation): Aromatic amino acid residues (UV). Reduced pyridine nucleotides (UV). Flavins (UV, blue). Chitin (broad). Chlorophyll (blue, green). General sources autluorescence: Dead cells (broad). Lipuscin (UV, blue). Cures: Long wavelength (also lower energy) light.[except 2-Phon] Avoid stress.

15 Phodamage Illumination energy not converted in emitted light (typ. <1%) or heat can enforce chemical reactions.

16 Recognise damaged cells Cells detach. Blebs form. Michondria swell. Cells do not make it through misis. Necrosis, Apopsis

17 Jens Jens Rietdorf: Rietdorf: Different Different microscopy microscopy techniques techniques will will be be discussed discussed which which very very light light efficient efficient avoid avoid phodamage phodamage discussed discussed in in following following Avoid phodamage Use decent dyes. Optimise illumination detection: Filtersets Detecrs Resolution (xy,z,t,intensity value, channels) Make use image processing ( deconvolution ). Add antioxidants (Trolox, ascorbic acid 2mg/ml) Use appropriate microscope techniques.

18 Contents Environment Physical integrity Attachment Temperature Gases (CO 2, O 2,H 2 O), ph. Osmolarity Illumination Autluorescence Phodamage Microscopy image processing techniques deconvolution Live cell microscopes (Time lapse sequence analysis)

19 Wide-field microscopy + deconvolution -Use a priori knowledge improve image quality -deconvolution is possible for all image dimensions : Along optical axis, time-lapse, color

20 Jens Jens Rietdorf: Rietdorf: Deconvolution Deconvolution can can increase increase signal--noise signal--noise ratio ratio reby reby allows allows reduction reduction excitation excitation light. light. Always Always use use deconvolution deconvolution before before estimating estimating how how much much light light has has be be put put in in sample sample reveal reveal relevant relevant information. information. deconvolution

21 Jens Jens Rietdorf: Rietdorf: Spinning Spinning disc disc confocals confocals a a good good alternative alternative single single beam beam scanning scanning confocals confocals as as y y use use very very low low excitation excitation light light intensities. intensities. Example Yokogawa unit

22 Jens Jens Rietdorf: Rietdorf: TIRF TIRF microscopy microscopy limits limits excitation excitation a a small small a a close close coverglas coverglas provides provides excellent excellent contrast. contrast. Total internal reflection fluorescence microscopy TIRFM prismless system

23 Example membrane fusion in TIRF

24 Jens Jens Rietdorf: Rietdorf: Example Example single single molecule molecule TIRF. TIRF. Example Pascale

25 Jens Jens Rietdorf: Rietdorf: Differently Differently shaped shaped structures structures may may be be labeled labeled with with same same dye dye still still saparable saparable in in different different channels channels by by object object detection detection approaches. approaches. Double Double exposure exposure for for different different fluorophores fluorophores can can be be avoided. avoided. Simultaneous multichannels I

26 scope Simultaneous multichannels II microimager camera unmix

27 Jens Jens Rietdorf: Rietdorf: Different Different microscopy microscopy methods methods more more or or less less suited suited for for different different applications. applications. Mark Mark 1=good. 1=good. A A very very rough rough estimate estimate made made emphasize emphasize pros pros cons cons different different methods methods with with respect respect live live cell cell imaging. imaging. Comparision ols Widefield (+deco) Confocal Light Depth Acquisition Volume Timelapse Flexibility efficiency discrimination speed imaging imaging Multibeam confocal 2-Phon TIRF (n.p) 1 5

28 Conclusions Keep environment constant convenient Use powerful dyes Think about resolution required (xy,z,t,intensity value, channels) minimize phostress Use appropriate microscopy method Use deconvolution

29 People involved ALMF: Rainer Pepperkok Timo Zimmermann Andreas Girod Kota Miura