BasicCoursePhDProg - Peter Evennett - schedule October 2014

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PhD Program: Basics of Light Microscopy - Peter Evennett / LMF - Course October 2014

SETUP - Thu 09th and Fri 10th October

Prepare day boxes and teaching stations (microscopes with cameras etc) on tables in LMF hallway on Thu
Setup of all needed course material in Galeria on Fri starting 9AM

Equipment needed

  • Microscopes (6 teaching stations, 1 demo station, Peter's diffraction demo scope)
  • Optical bench
  • Spinning disk gadget
  • Polarizing sheets
  • Diffraction Grids
  • Sample map for each teaching station with: diatomes, liver tissue, Convolaria, Benzocain crystals
  • microscope handout booklet for each teaching station
  • First day handout for students
  • Light torches
  • Cameras and labtops for teaching stations
  • Cleaning stations for each system containing: cover slips, slides, Qtips, water, 70% EtOH, lens cleaning tissue, EDTA, immersion oil, nail polish
  • Waste and glass waste for each teaching station
  • Box with screw drivers (1x 3mm, 2x 1.5mm), telescope, eye pieces, microscope ruler, mirror slide, DIC objective and Wollaston prism for each microscope
  • 2 small polarization filter sheets for each microscope
  • Box with red, green, blue, black board marker, pen, pencil, sharpener, ruler, calculator for each microscope

DAY ONE - Mon 13th October

FROM TO ACTIVITY TOPICS Responsible person(s) Materials needed
8:30 09:00 FINAL PREP Test everything works BioDIP TEAM
9:00 9:35 INTRODUCTION COURSE INTRO
WHO is WHO? Round table
BioDIP TEAM White board and pen; ask students for volunteering for Friday's project discussion round
9:35 10:30 Session 1
Theory
PRINCIPLES OF LIGHT MICROSCOPY
Historical aspects
Very basic components and principles (incl. refraction and resolution basics)
PETER EVENNETT (BioDIP TEAM) students shortly watch airy pattern from hole in mirror at the teaching microsopes (~10:08 - 10:15)
10:30 10:45 BREAK BREAK
10:45 11:28 SESSION 1
Practice
3 groups, 12min Rotation (+3min for switching stations)
Timer: SEBASTIAN
capillary/plastic (Huisken) in oil demo
Coin-in-cup demo
red, green laser through water bath -
measure angles, calculate refraction;
show immersion objectives
measure refraction with modern refractometer
JAN
BRITTA
DAVIDE
*capillaries, oil
* coin-in-cup, water
* red/green laser pointer with holder stand, plastic refraction demo setup with protractor and water, for next time: prepare laminated image of ice bear in water/air
*water, glycerol, oil immersion objectives
*refractometer
11:28 12:12 SESSION 1
Theory
Introduction to lenses and objectives
Numerical aperture
Magnification
PETER EVENNETT cut objectives
12:12 13:00 LUNCH LUNCH
13:00 13:45 SESSION 1
Practice
15min (!) Rotation
Timer: Sebastian
Milky Glass Block demo
Show and discuss microscope parts (upright and inverted)
NA measurements
JAN
BRITTA
BERT
*milky glass block, teaching microscope, coloured filters for microscope, iris objective
*slides with arrows, simple upright and inverted microscope
*horizontal protractor on stand, objective holder, 2 air objective with different (!) NAs (and maybe an iris objective), calculator, pencils, rubber
13:45 14:30 SESSION 2
Theory
Microscope illumination
Conjugate planes
Apertures
PETER EVENNETT
14:30 14:45 BREAK BREAK
14:45 15:55 SESSION 2
Practice
(30min Rotation)
Timer: SEBASTIAN
conjugate planes on Peters microscope
conjugate planes on optical bench
PETER EVENNETT
JAN
*Peters microscope setup
*optical bench demonstrating a transmitted light microscope, incl. light source
conjugate planes scheme hand outs
15:55 16:30 SESSION 3
Theory
Koehler illumination
Epi illumination
(Microscope alignment)
PETER EVENNETT
16:30 17:30 SESSION 3
Practice
Koehler illumination
(Microscope alignment)
BioDIP TEAM students manual microscopes, screwdrivers, nice brightfield sample
17:30 18:00 SESSION 4
Theory & Practice
Eyepieces
Depth of field
Depth of focus
PETER EVENNETT
BioDIP TEAM
students microscopes with 10x/0.25 and 40x/0.65 objectives
Stereoscope with a "sample"

DAY TWO - Tue 14th October

FROM TO ACTIVITY TOPICS Responsible person(s) Materials needed
9:00 10:10 Recap day 1 BioDIP TEAM Questions
teaching microscopes; conjugate planes schemes
10:10 11:05 SESSION 1a
DEMONSTRATION
+ THEORY
Diffraction and Diffraction slide fun
Diffraction string show
Diffraction and Resolution
PETER EVENNETT diffraction slides, torch, "dashed" string, fabric, funny glasses
11:05 11:20 BREAK COFFEE BREAK


11:20 11:50 SESSION 1b
Demo and Theory
ABBE's diffraction demonstration BERT Dan's ABBE Diffraction demo setup
11:50 12:20 SESSION 1b
Practice
Diffraction hands on on student microscopes BioDIP TEAM gratings, place holder for objective Nomarski prism, piece of card board, microscopes without condenser, closed field diaphragm, some small scissors would be nice
12:20 13:00 SESSION 1b
Demo and Theory
Peter's Diffraction video (w/o parts on Dark field and Phase contrast!) JAN
(PETER EVENNETT)
video
13:00 14:00 BREAK LUNCH
14:00 14:15 SESSION 1b
Theory
Wrap - up & Q & A
Diffraction
PETER EVENNETT
JAN
14:15 15:10 SESSION 2
Theory and Demo
Lens abERRations and corrections
objective labeling/reading
PETER EVENNETT
JAN
lenses with various sizes and shapes
magnetic lens + laser suitcase on white board
(15:05 - 15:10)
15:10 15:15 SHORT BREAK FRESH AIR
15:15 15:45 SESSION 3
Theory
Introduction to contrast PETER EVENNETT
15:45 16:00 BREAK COFFEE BREAK
16:00 16:10 SESSION 3a
Video
Peter's Dark Field diffraction video part JAN
(PETER EVENNETT)
video
16:10 16:20 SESSION 3a
Theory
Dark Field microscopy PETER EVENNETT Zeiss reflection dark field condenser from W3 + new 100x iris objective
16:20 16:50 SESSION 3a
Practice
Dark Field microscopy BioDIP TEAM diatom slides; cells/Chlamy's from Gaia; samples out of Ruth's Hay infusion
16:50 17:00 SESSION 3b
Video
Peter's Phase contrast diffraction video part JAN
(PETER EVENNETT)
video
17:00 17:20 SESSION 3b
Theory
Basics of Phase Contrast microscopy PETER EVENNETT
17:20 17:25 SESSION 3b
Demonstration
Cheek cell prep JAN Q-tips, slides, cover slips, PBS, plastic pipette, nail polish
17:25 18:10 SESSION 3b
Practice
Phase Contrast microscopy BioDIP TEAM cheek cell samples
19:00 open end Dinner with Peter BioDIP TEAM good mood, time

DAY THREE - Wed 15th October

FROM TO ACTIVITY TOPICS Responsible person(s) Materials needed
9:00 10:10 Recap day 2 BioDIP TEAM example you tube videos on dark field and phase contrast (9:05-9:10)
Questions, pair of sieves, torches, microscopes
10:10 10:45 SESSION 1
Theory
Objective reading
lateral vs angular magnification
PETER EVENNETT
JAN
objectives, eyepieces, overhead projector
10:45 11:00 BREAK BREAK BREAK
11:00 12:10 SESSION 1
Practice
Group rotation
(30min each)
Cover glasses, sample mounting, Objective cleaning and
infinity optics bench demo
Objective reading
JAN

BRITTA
Coverglasses (High preciscion), different oils, slides, different cleaning reagents, cover glass thickness measure meter
bench demo of infinity optics
box with broken objectives + two 160 tube lens objectives
12:10 13:00 SESSION 2
Theory
Polarized light microscopy PETER EVENNETT for students and Peter: calcite crystal blocks, polarizers, paper with black spot;
overhead projector
13:00 14:00 BREAK LUNCH
14:00 14:05 SESSION
Meet & Greet
Meeting Jean-Marc Verbavatz, Leader EM facility JEAN-MARC
(JAN)
Q & A with Jean-Marc about his facility and cooperation with LMF
14:05 14:45 SESSION 2
Practice
Polarized light microscopy PETER EVENNETT
BioDIP TEAM
overhead projector, hair, stone samples, benzocain crystals, rulers, gummy bears, plastic dishes, ...
14:45 15:10 SESSION 3
Theory
Differential Interference Contrast (DIC) PETER EVENNETT
15:10 15:55 SESSION 3
Practice
Differential Interference Contrast (DIC) BioDIP TEAM
JAN
cheek cell samples, diatoms
15:55 16:10 BREAK BREAK BREAK
16:10 16:30 Question round Questions to Peter Evennett PETER EVENNETT
BioDIP TEAM
questions to Peter;
DIC video from web (16:15 - 16:20)
remind students on Friday's project discussion round
16:30 17:05 SESSION 3
Theory
Fundamental concepts of fluorescence JAN
BRITTA
fluorescent liquids in bottles plus laser pointer (blue, green, red; need stronger red + green ones!)
17:05 18:00 SESSION 4
Theory
Introduction to fluorescence microscopy
and light sources
DAVIDE
JAN
• Light sources
• Lamp houses

DAY FOUR - Thu 16th October

FROM TO ACTIVITY TOPICS Responsible person(s) DEMO Activities Materials needed
9:00 10:10 Recap day 3 BioDIP TEAM - Questions
10:10 10:50 SESSION 1
Theory
2nd part Fluorescence Microscopy DAVIDE
JAN
- Spectra
Filters
Filter cubes
10:50 11:05 SESSION 1
Practice
Filters BioDIP TEAM - laminated sheets with grids for spectra drawing
red, green, blue, black board markers
EtOH spray bottles, tissue
11:05 11:20 BREAK BREAK BREAK BREAK BREAK
11:20 12:25 SESSION 1
Practice
Spectraviewer
Aligment of Mercury arc lamps
Fluorescence imaging at microscopes
JAN
BioDIP TEAM
- lamp house, screw driver
laminated sheets with DAPI/FITC/TRITC Spectra
red, green, blue, board markers
EtOH spray bottles + tissue
Fluorescent labeled samples (Convallaria)
12:25 12:35 SESSION 1
Theory
Chromatic Aberration Correction DAVIDE -
12:35 13:15 SESSION 2
Theory
Detectors BRITTA - CCD chip paper weight
Demo: CCD chip under stereo microscope
example cameras showing different chip sizes
13:15 14:00 BREAK LUNCH LUNCH LUNCH LUNCH
14:00 14:30 SESSION 2
Demo & Theory
Detectors BRITTA bench show
advacned detectors lecture
RT spot with RGB slider and camera objective
PC
nice sample for binning (coffee cup)
14:30 14:35 SESSION 2
Practice
Detectors
"QE" show
JAN students count times of laser pointer blinking blinking laser pointer
14:35 15:00 SESSION 3a
Theory & Demo
Digital images BERT
JAN
Nyquist-Shannon
what is a pixel
spatial calibration
overhead projector
sheets with different sized squares as example for dexel size
gummi bears, Sesame seeds
15:00 15:35 SESSION 3
Practice
calculating dexel/pixel size BioDIP TEAM practice calculating needed dexel size for given optical setup calculator
text with given optical setup
15:35 15:50 SESSION 3b
Theory
Quantitative Imaging BERT digitization in space, time & intensity
aliasing
-
15:50 16:05 BREAK BREAK
16:05 17:15 SESSION 4
Practice
discussion and practice imaging with CCD/CMOS cameras on student microscopes BioDIP TEAM check out basic camera vocabulary
discussing spec sheets of student microscope cameras
spatial calibration with ruler and FIJI
LUT, saturation, histogram etc
camera spec sheets
Hamamatsu camera comparison list
camera vocabulary checklist
microscope ruler
Fluorescence Sample slides (Kidney / Cells...)
17:15 17:20 SESSION
Meet & Greet
Meeting Marc Bickle, leader of Screening facility MARC
(JAN)
Q & A with Marc about his facility and cooperation with LMF
17:20 18:20ish
open end
SESSION 4+5
Practice
Theory/Tour
finish practice discussion form before
Tour through LMF
LMF TEAM first show to whole group:
BioDIP Wiki with pages for all systesms
ask participants which systems they want to see

DAY FIVE - Fri 17th October

FROM TO ACTIVITY TOPICS Responsible person(s) Materials needed
9:00 10:00 Recap day 4 BioDIP TEAM Question sheets with the real case problem
calculators, pens
10:00 10:30 SESSION 1a
Theory
OPTICAL SECTIONING
introduction
widefield & deconvolution
chromatic aberration & correction
JAN labtop
10:30 10:45 BREAK BREAK BREAK
10:45 11:40 SESSION 1b
Theory & Demo
OPTICAL SECTIONING
Laser Scanning Confocal
2PM
JAN
SEBASTIAN
"confocal equipment":
laser pointer, mirror, broken scanning mirror
dual laser pointer (blue, green, red) to demonstrate penetration of diff. wavelength
11:40 12:25 SESSION 1c
Theory & Demo
OPTICAL SECTIONING
Spinning disc confocal
TIRF
SPIM
JAN
BRITTA
DAVIDE
* DAN's "spinning-disc-on-a-bench"
* small glass cube with olive oil over milky water (use more oil than water!) plus Dan's blue laser pointer
* glass block with brain/scull and red light sheet laser from ZEISS
12:25 12:35 SESSION 1c
Demo
OPTICAL SECTIONING
SPIM
PETE PETE's SPIM-in-a-suitcase
12:35 12:45 SESSION 1d
Theory
OPTICAL SECTIONING
Apotome
JAN
12:45 13:30 LUNCH LUNCH LUNCH Optional: LMF tour
13:30 13:45 SESSION 1e
Theory
OPTICAL SECTIONING
final remarks
JAN
13:45 14:10 SESSION 1f
Theory
SUPER-RESOLUTION
SIM
STORM
BERT
14:10 14:20 SESSION 2 Planning your Imaging Experiment DAVIDE + introduction to new user project questions
14:20 15:00 SESSION 3
Project discussion
Two volunteers present their imaging problems shortly and discuss it with the whole group BioDIP TEAM
+ BENOIT
NOTE: ask for volunteers already on Monday, than again on Wednesday;
15:00 16:00 SESSION 3 Course evaluation BioDIP TEAM Gummy bear bags and/or fruits
white board + marker
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