Michael Hooker Microscopy Facility (MHMF.ORG)
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Michael Hooker Microscopy Facility (MHMF.ORG) |
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Light Microscopy for Biomedical Research - Lab exercises
Day 1 -- Parts of a
microscope, care, basic digital acquisition
1:30-3pm
- Exercise handout - Condenser Aperture open/closed (save images), compare image quality, color balance, Jello, (if time DIC)
- Stations
- Leica DMIRB
- diagram (print version)
- user manuals: setup Kohler digital acquisition
- Nikon Microphot upright
- diagram (print version)
- user manual: MultiMode (incl. kohler and digital acquisition)
- Nikon TE2K Spinning Disk
- diagram (print version)
- user manual Spinning Disk-widefield (incl. kohler and digital acquisition)
- Samples
- H&E slides
- Diatoms
- Exercises:
- Guided tour - parts of the microscope - follow transmitted light path - camera(s) (10 mins)
- Kohler illumination H&E (30 mins)
- Condenser aperture effects - thin card view - jello block - (10 mins)
- Dry lens corrections - adjust Cheek cells| DMIRB | MM | SDC (15 mins)
- DIC ? Cheek cells | DMIRB | MM | SDC (20 mins)
- Phase ? Cheek cells | MM | SDC (10 mins)
- Color H&E | DMIRB | HOOKE (10 mins)
- Care of scope - gentle - no drop objectives - no scrub lens- etc. - cleaning (15 mins)
Day 2
D2 Morning (11am-lunch): Kohler review, DIC and fluorescence
- Exercise handout - 1. Cheek DIC quick. 2. Fluorescence with FM 1-43, 4. Sub resolution beads + big ones
- Stations
- Leica DMIRB
- Nikon TE2K MultiMode
- Nikon TE2K Spinning Disk
- Samples
- Cheek cells + FM 1-43
- slides
- coverslips
- pipet tips + pipetman
- FM solution
- cone of light
- Molec. Probes BPAE slides
- pollen grains
- sub-resolution beads
- Exercises:
- Guided tour of light path from arc lamp to camera/eye (5 mins)
- Kohler for fluorescence | DMIRB | MM | SDC (5 mins)
- Changing arc lamp bulbs + alignment (In talk 5 mins)
- Basic single channel 10x dry , 40x dry versus 40x oil intensity = NA4/mag2 (15 mins)
- Two+ channels Probes slides or Pollen grain (10 mins)
- DAPI long pass cube or FITC Long pass or walk to 510 - see red and green edges due to cross excitation (10 mins) (which systems?)
- Sub-resolution beads - demonstrates light house in fog (5 mins)
- S/n exposure time (adjust electronic gain) versus noise (5 mins)
- Binning to get back photons
- Finish above in afternoon
D2 Afternoon (2pm-end): Time lapse & DIC review(?)
- Exercise handout
- NEED TIME LAPSE ACQUISITION INSTRUCTIONS!!! (Same for all systems)
- Stations
- Leica DMIRB
- Nikon TE2K MultiMode
- Nikon TE2K Spinning Disk
- Samples:
- SOMETHING THAT MOVES IN 2-D - beads Brownian motion (slow down in glycerol)
- Trans
- Fluorescence
- 90% glycerol
- transfer pipets
- Arabidopsis + FM 4-64 (?)
- Cardiac myocytes in 6 well plastic + Fluo-4 (?)
- Excercises
- Time lapse
- finish anything from the morning
Day 3
D3 Morning(10:45a-lunch): Confocal I: Sensitivity, pinhole, filters
- Exercise handout
- Stations
- Zeiss 510 Meta
- Leica SP2
- Leica SPE
- Samples
- Molecular Probes tissue slides
- Exercises:
- Tissue section slides Molec Probes
- By eye 1st - do Kohler setup
- And setup DIC
- Remember epi fluorescence view
- The confocal startup - let users run but lots of guidance from instructors
- Arc lamp on - Laser on - power
- Filter configuration
- AOTF - Detectors
- Scan etc, etc, etc, etc, etc, etc, etc, etc, etc, etc, etc, etc, etc, etc,
- Note image versus eyeball view, scan is delayed, see less material, but finer detail, "out of focus" view,
- Now turn on transmitted or DIC detection.
- Averaging, scan speed,
- Bleaching (?) FM 1-43 cheek cells
- Setting brightness, gain versus averaging, black clipping, overload
D3 Afternoon1 (1-3p): Confocal II: Z-series, time-lapse, spectral imaging
**images from these exercises will be used for analysis exercises later**
- Exercise handout
- Stations
- Zeiss 510 Meta
- Leica SP2
- Leica SPE
- Samples/Exercises:
- Z-series: pollen grain slides
- Time series: Arabidopsis + FM
- mustard seed rootlets
- FM solution
- slides
- coverslips
- media for plants
- pipette tips + pipetman
D3 Afternoon2 (3p-end): 3-D rendering
- Exercise handout
- Stations
- Volocity on Snell
- user guide
- particle tracking
- Zeiss software on Brewster
- user guide
- Zeiss software on 510 acquisition station
- user guide
- Samples
- Images from Confocal II exercises
Day 4
D4 Morning (11a-lunch): Digital image handling: calibration, contrast, time-lapse, averaging/exposure
- Exercise handout
- Stations
- Leica DMIRB
- Nikon TE2K MultiMode
- Nikon TE2K Spinning Disk
- Exercises:
- Wide field - images provided (all computer activites!)
- Intensity scaling with 8 bit versus 16 bit.
- Binning
- Distance calibration
- Contrast - auto versus manual in photoshop
- Make movies, export, reassemble, compression
- Power point
D4 Afternoon (2-3:15p): Confocal Multi-Channel
- Exercise handout
- Zeiss 510 Meta
- Leica SP2
- Leica SPE
- Samples
- Molecular Probes slides
- (?) Cheek cells with Calcein green + MitotrackerRed
- slides
- coverslips
- pipet tips + pipetman
- calcein green solution
- mitotraker red solution
- Exercises:
- Confocal III (1 hr)
- Multi-channel - simultaneous versus sequential
- Get 2-channel z-series for afternoon computer exercise
? when will they do the 3-D for this?- Spectral imaging: Acridine Orange (change vesicle pH with NH4Cl?)
- FRAP -- Kristin's bugs
Day 5 --
Data handling/Image processing - movies, scaling, compression, resizing,
conversion, format for Power Point
12:30-2pm
- Exercise handout
- Brewster
- Snell
- Nipkow?
- Samples:
- Use files from the past week
- Exercises:
- Record camera dark noise - long exposure/gain
- Record readout noise short exposure high gain
- Measure camera noise
- RMS calculation
- Fluorograms - on already acquired images
- Color conversion - monochrome
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