BasicCoursePhDProg - Peter Evennett - schedule April 2016

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PhD Program: Basics of Light Microscopy - Peter Evennett / LMF - Course April 2016

Tentative Schedule

SETUP - Wed and Thu April (Fri TA course)

Prepare day boxes and teaching stations (microscopes with cameras etc) on tables in LMF hallway on Thu
Part of teaching microscopes where already set up before for the TA course
Setup of all needed course material in Galeria on Thu starting 11AM

Equipment needed

  • microphone for Peter E.
  • 10 tables (we need to order them early on - Davide will do; for Wed evening)
  • Microscopes (6 teaching stations, 1 demo station, Peter's diffraction demo scope) (we will try to order another microscope body)
  • cell culture microscope (normally with EM people; currently moved to cell culture room 4th floor south; Jan knows and will take care of getting it for the course)
  • stereo microscope with coin
  • Optical bench
  • Spinning disk gadget
  • Overhead projector with digital source switch
  • 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 lab-tops 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 18th April

FROM TO ACTIVITY TOPICS Responsible person(s) Materials needed
8:30 09:00 FINAL PREP Test everything works BioDIP TEAM
9:00 9:38 INTRODUCTION COURSE INTRO
WHO is WHO? Round table
BioDIP TEAM White board and pen; ask students for volunteering for Friday's project discussion round
(we need four volunteers)
9:38 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 (~10:04 - 10:10)
10:30 10:45 BREAK BREAK
10:45 11:25 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, beaker for water
* red/green laser pointer with holder stand, plastic refraction demo setup with protractor and water, laminated image of ice bear in water/air
*water, glycerol, oil immersion objectives
*refractometer
11:25 12:15 SESSION 1
Theory
Introduction to lenses and objectives
Numerical aperture
Magnification
PETER EVENNETT cut objectives
12:15 13:00 LUNCH LUNCH
13:00 13:50 SESSION 1
Practice
15min (!) Rotation
Timer: SEBASTIAN
Milky Glass Block demo
Show and discuss microscope parts (upright and inverted)
NA measurements
JAN/DAVIDE
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
13:50 14:40 SESSION 2
Theory
Microscope illumination
Conjugate planes
Apertures
PETER EVENNETT
14:40 14:55 BREAK BREAK
14:55 15:55 SESSION 2
Practice
(30min Rotation
inlc. time for switching)
Timer: SEBASTIAN
conjugate planes on Peters microscope
conjugate planes on optical bench +
effect of changing field and aperture diaphragms on demo scope
PETER EVENNETT
JAN
*Peters microscope setup
*optical bench demonstrating a transmitted light microscope, incl. light source
conjugate planes scheme hand outs
15:55 16:20 SESSION 3
Theory
Koehler illumination
(Microscope alignment)
PETER EVENNETT
16:20 17:20 SESSION 3
Practice
Koehler illumination
(Microscope alignment)
BioDIP TEAM students manual microscopes, screwdrivers, nice brightfield sample
17:20 17:45 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"
17:45 17:55 SESSION 3
Theory
Epi illumination PETER EVENNETT

DAY TWO - Tue 19th April

FROM TO ACTIVITY TOPICS Responsible person(s) Materials needed
9:00 10:00 Recap day 1 BioDIP TEAM Questions
teaching microscopes; conjugate planes schemes
10:00 10:05 Theory Introduction to
Peter Evennett's videos
Jan You tube videos
10:05 10:45 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
if available: wave/ripple tank; interferometer
10:45 11:00 BREAK COFFEE BREAK


11:00 11:25 SESSION 1b
Demo and Theory
ABBE's diffraction demonstration BERT Dan's ABBE Diffraction demo setup
11:25 12:00 SESSION 1b
Practice
Diffraction hands on on student microscopes BioDIP TEAM rulers as gratings, place holder for objective Nomarski prism, piece of card board, microscopes without condenser, closed field diaphragm, some small scissors would be nice
12:00 12:15 SESSION 1c
Theory
Wrap - up & Q & A
Diffraction
PETER EVENNETT
JAN
12:15 13:15 BREAK LUNCH
13:15 13:50 SESSION 2
Theory and Demo
Lens abERRations and corrections
finite versus infinite tube length
objective labeling/reading
PETER EVENNETT
JAN
lenses with various sizes and shapes
13:50 14:05 SESSION 3
Theory
Objective reading
lateral vs angular magnification
PETER EVENNETT
JAN
objectives, eyepieces, overhead projector with digital source switch
14:05 14:10 SESSION 2
Demo
demo of spherical aberration JAN magnetic lens + laser suitcase on white board
14:10 14:50 SESSION 3
Practice Round 1
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
cell culture microscope with finite tube length
14:50 15:25 SESSION 3
Practice Round 2
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
cell culture microscope with finite tube length
15:25 15:40 BREAK COFFEE BREAK
15:40 16:00 SESSION 4
Theory
Introduction to contrast BERT
16:00 16:25 SESSION 5a
Theory
Basics of Dark Field microscopy JAN Zeiss reflection dark field condenser from W3 + new 100x iris objective
video with dark field example; from web and own
16:25 17:00 SESSION 5b
Practice
Dark Field microscopy BioDIP TEAM diatom slides
17:00 17:30 SESSION 6a
Theory
Basics of Phase Contrast microscopy DAVIDE including videos from web and own
17:30 17:35 SESSION 7
Demonstration
Cheek cell prep JAN Q-tips, slides, cover slips, PBS, plastic pipette, nail polish
17:35 18:00 SESSION 6b
Practice
Phase Contrast microscopy BioDIP TEAM diatomes and similar samples
cell culture microscope with cells in a dish as live example for phase contrast

DAY THREE - Wed 20th April

FROM TO ACTIVITY TOPICS Responsible person(s) Materials needed
9:00 10:05 Recap day 2 BioDIP TEAM Questions, pair of sieves, torches, microscopes
10:05 10:30 SESSION 1
Demonstration
Polarized light microscopy SEBASTIAN
JAN
for students and Sebastian: calcite crystal blocks, polarizers, paper with black spot;
overhead projector
stone samples, gummy bears, plastic dishes, plastic rulers...
10:30 11:10 SESSION 1
Theory
Polarized light microscopy SEBASTIAN info about LC-PolScope from openpolscope.org/
introduce responsible person for Brugues' PolScope
11:10 11:25 BREAK BREAK BREAK
11:25 11:45 SESSION 1
Practice
Polarized light microscopy BioDIP TEAM hair, stone samples, benzocain crystals, tissue pieces ...
11:45 12:22 SESSION 2
Theory
Differential Interference Contrast (DIC) BRITTA
12:22 13:00 SESSION 2
Practice
Differential Interference Contrast (DIC) BioDIP TEAM
JAN
DIC-objectives with respective Nomarski prisms;
cheek cell samples, diatoms
13:00 14:00 BREAK LUNCH
14:00 14:05 SESSION
Meet & Greet
Meeting Mark Bickle, Leader Technology development studio
screening facility
MARK
(JAN)
Q & A with Mark about his facility
14:05 14:25 Wrap-up
compare methods
Transmitted light contrasting techniques BERT
BioDIP TEAM
first comparison than
Quiz with various videos of the different techniques; remind students on Friday's project discussion round
14:25 14:30 QUESTION ROUND questions to Peter Evennett PETER EVENNETT
JAN
14:30 14:57 SESSION 3
Theory
Fundamental concepts of fluorescence JAN fluorescent liquids in bottles plus laser pointer (blue, green, red; need stronger red + green ones!)
(in seminar room for darkness)
14:57 15:35 SESSION 4a
Theory + Demo
Introduction to fluorescence microscopes
and light sources
DAVIDE
JAN
• Light sources
• Lamp houses
15:35 15:50 BREAK BREAK BREAK
15:50 16:10 SESSION 4b
Theory
Introduction to fluorescence microscopy
Filters, spectra etc.
DAVIDE
JAN
Spectra
Filters
Filter cubes
16:10 16:35 SESSION 4c
Practice
Filters BioDIP TEAM laminated sheets with grids for spectra drawing
red, green, blue, black board markers
EtOH spray bottles, tissue
16:35 16:50 SESSION 4d
Theory and Demo
Introduction to fluorescence microscopy
Filters, spectra
objective transmission curves
DAVIDE spectra viewer
16:50 18:00 SESSION 4e
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 (Convalaria)
19:00 open end Dinner with Peter BioDIP TEAM good mood, time

DAY FOUR - Thu 21st April

FROM TO ACTIVITY TOPICS Responsible person(s) DEMO Activities Materials needed
9:00 10:00 Recap day 3 BioDIP TEAM - Questions (need to be adjusted to new course scheme!)
10:00 10:15 SESSION 1
Theory
Fluorophores HELLA -
10:15 10:45 SESSION 2
Theory
Detectors BRITTA - CCD chip paper weight
Demo: CCD chip under stereo microscope
example cameras showing different chip sizes
10:45 11:00 BREAK BREAK BREAK BREAK BREAK
11:00 11:25 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)
11:25 11:40 SESSION 2
Practice
Detectors
"QE" show
JAN students count times of laser pointer blinking blinking laser pointer
11:40 11:55 SESSION 3a
Theory
Digital images BERT Nyquist-Shannon
what is a pixel
spatial calibration
11:55 13:00 BREAK LUNCH LUNCH LUNCH LUNCH
13:00 13:05 SESSION
Meet & Greet
Meeting Tobias Fürstenhaupt, Leader EM facility Tobias
(JAN)
Q & A with Tobias about his facility and cooperation with LMF -
13:05 13:10 SESSION 3a
Demo
Digital images JAN Nyquist-Shannon
spatial calibration
overhead projector
sheets with different sized squares as example for dexel size
gummi bears, Sesame seeds
13:10 13:35 SESSION 3a
Practice
calculating dexel/pixel size BioDIP TEAM practice calculating needed dexel size for given optical setup calculator
text with given optical setup (within Bert's presentation)
13:35 13:45 SESSION 3a
Practice
calculating dexel/pixel size BERT practice calculating needed dexel size for given optical setup evaluation of the results from practical plus more calculation examples
13:45 13:55 SESSION 3b
Theory
Quantitative Imaging BERT digitization in space, time & intensity
aliasing
-
13:55 15:10 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 (in microscope handbook at each teaching scope)
Hamamatsu camera comparison list (extra sheet)
camera vocabulary checklist (in microscope handbook at each teaching scope)
microscope ruler
Fluorescence Sample slides (Kidney / Cells...)
15:10 15:25 BREAK BREAK
15:25 15:30 SESSION
Meet & Greet
Meeting Hella Hartmann, BioDIP Coordinator HELLA
(JAN)
Q & A with Hella about BioDIP and cooperation between the participating facilities BioDIP website
15:30 15:43 SESSION 5a
Theory
OPTICAL SECTIONING
introduction
widefield & deconvolution
JAN - labtop
15:43 15:53 SESSION 6
Theory
CHROMATIC ABERRATION DAVIDE
15:53 16:20 SESSION 5b
Theory & Demo
OPTICAL SECTIONING
Laser Scanning Confocal
JAN "confocal equipment":
laser pointer, mirror, broken scanning mirror
16:20 16:40 BREAK BREAK
16:40 17:15 SESSION 5c
Theory& Demo
OPTICAL SECTIONING
2-Photon Microscopy
SEBASTIAN
JAN
dual laser pointer (blue, green, red) to demonstrate penetration of diff. wavelength
17:15 17:45 SESSION 5d
Theory
OPTICAL SECTIONING
Spinning Disc Confocal
BRITTA
JAN
beer bottle from Plzen
laminated sheets and copies of Nipkow's and Petran's patents
17:45 18:00 SESSION 5d
Demo
OPTICAL SECTIONING
Spinning Disc Confocal
JAN DAN's "spinning-disc-on-a-bench"

DAY FIVE - Fri 22nd April

FROM TO ACTIVITY TOPICS Responsible person(s) Materials needed
9:00 10:10 Recap day 4 BioDIP TEAM Question sheets with the real case problem
calculators, pens
voting kit?
10:10 10:25 SESSION 1a
Theory & Demo
OPTICAL SECTIONING
TIRF
BRITTA small glass cube with olive oil over milky water (use more oil than water!) plus Dan's blue laser pointer
10:25 10:50 SESSION 1b
Theory & Demo
OPTICAL SECTIONING
SPIM
DAVIDE glass block with brain/scull and red light sheet laser from ZEISS
10:50 11:05 BREAK BREAK
11:05 11:15 SESSION 1c
Demo
OPTICAL SECTIONING
SPIM
CHRISTOPHER PETE's SPIM-in-a-suitcase
11:15 11:22 SESSION 1d
Theory
OPTICAL SECTIONING
Apotome
JAN
11:22 11:30 SESSION 1e
Theory
OPTICAL SECTIONING
final remarks
JAN
11:30 12:05 SESSION 2
Theory
SUPER-RESOLUTION
SIM
STORM
STED
Airyscan
Sample Preparation
BERT
12:05 12:15 DISCUSSION preparation for Monday hands-on sessions SEBASTIAN
BioDIP team
white board
12:05 13:15 LUNCH LUNCH LUNCH
13:15 13:22 SESSION 3 Planning your Imaging Experiment DAVIDE + introduction to new user project questions
13:22 13:32 SESSION
Meet & Greet
Meeting Alf Honigmann, STED-lab ALF
(JAN)
Q & A with Alf about his lab, STED, and cooperation possibilities
13:32 13:52 SESSION
Meet & Greet
Meeting Benoit Lombardot, Leader Scientific Computing facility BENOIT
(JAN)
introduction to bioinformatics service and image processing facility
13:52 14:45 SESSION 4
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
14:45 15:45 SESSION 6 Course evaluation BioDIP TEAM Gummy bear bags and/or fruits
white board + marker

DAY SIX - optional - Mon 25th April afternoon

FROM TO ACTIVITY TOPICS Responsible person(s) Materials needed
12:00 13:00 LUNCH LUNCH LUNCH
13:00 13:45 Preparation for hands-on OPTICAL SECTIONING / SUPER-RESOLUTION BioDIP team MZ1, LZ2, CZ6, SD4, D4, H1, H2, W1
we need to decide which systems to use
13:45 14:00 Meeting students
at switchboard
OPTICAL SECTIONING / SUPER-RESOLUTION BioDIP team sign-up sheets for both hands-on sessions
students decide which two systems (one per session) they want to get to know
distribute students into groups
14:00 15:30 SESSION I
hands-on
OPTICAL SECTIONING / SUPER-RESOLUTION BioDIP team MZ1, LZ2, CZ6, SD4, D4, H1, H2, W1
15:30 16:00 BREAK COFFEE KATJA
16:00 17:30 SESSION II
hands-on
OPTICAL SECTIONING / SUPER-RESOLUTION BioDIP TEAM MZ1, LZ2, CZ6, SD4, H1, H2, W1

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