8.30 Assignments

ASSIGNMENT 10:
For the advanced and courageous – this assignment is rather demanding.

A. Please look first at the web pages (links) below, where you can find basic information on cell microscopy.

Electron Microscope Tomography
http://www.lbl.gov/Science-Articles/Archive/sabl/2006/Jan/01-resolution-gap.html
http://www.fei.com/applications/biology-life-sciences.aspx?source=google&cmp=NanoB&ag=gen">
http://jcb.rupress.org/cgi/content/full/153/6/F25
http://en.wikipedia.org/wiki/Electron_microscope
http://en.wikipedia.org/wiki/Saccharomyces_cerevisiae

Useful links – Confocal Microscopy
http://en.wikipedia.org/wiki/Confocal_microscopy
http://depts.washington.edu/keck/intro.htm
http://www.loci.wisc.edu/confocal/confocal.html

Microtome
http://en.wikipedia.org/wiki/Ultramicrotome

Laminar box
http://en.wikipedia.org/wiki/Laminar_flow_cabinet

B. Download the Training B Roll file (in case your connection allows it – this file is 113 MB). Here, you´ll find footage filmed at a research institute where they examine cells via different microscopes. Please view the video and compare these shots with what you have read. The script will help you to do it. Then think about the shots you would like to use, in what order you would put them and what commentary you would use.

Video:

Laboratory with electron microscope tomography

• 00:00 Grid with a sample is put into a holder (several tries), D
• 01:01 Knocking-up the holder to place the grid the right way, D
• 01:10 Detail of the holder with the grid, D
• 01:20 Another try – placing the grid into a holder, tracking in shot, D
• 01 43 Taking a stick and fixation of a grid, D
• 01 58 Scientist A choosing a sample, D
• 02 15 Scientist A puts a grid into a holder (his face in background), MS
• 02 30 Scientist A takes a stick, fixing the grid, takes out the holder, D
• 03 02 Taking the grid with the sample from a sample plate D,
• 03 34 Taking the grid with the sample from a sample plate – second take, D
• 03 53 Scientist A before the microscope takes out the holder, MLS
• 04 12 Scientist A before the microscope takes out the holder and places it into the microscope
• 04 39 Scientist A places the holder into microscope – reverse angle, MLS
• 04 59 Pushing the holder into microscope, D
• 05 08 Placing the holder into microscope MLS, reverse angle
• 05 32 Clicking on a mouse, displays in background, MS
• 05 45 Scientist A looking into microscope, turning the buttons, MLS
• 05 53 Left hand turning buttons, D
• 06 01 Scientist A looking into microscope, MLS
• 06 22 Face of a scientist A looking into microscope, MS
• 06 29 Right hand gripping controller, D
• 06 35 Scientist A looking into microscope, MLS, reverse angle
• 06 44 Face of a scientist A, MS
• 07 05 Right hand manipulating with mouse, pan up to face, tracking in shot
• 07 20 Scientist A in front of computer displays, MLS
• 07 29 Scientist A moving from computer to microscope on the same rolling-chair, MLS, tracking his movements
• 07 50 Section of a cell on a computer display, D, pan up to the face
• 08 17 Face of a scientist A, D
• 08 27 Cells on a display from behind the scientist A, MS, pictures of cells are changing
• 09 00 Scientist A in front of a computer, reverse angle, MLS
• 09 23 Scientist A looking into the microscope, reverse angle, MLS
• 09 31 Left hand manipulating, D
• 09 42 Scientist A shifts aside oculars, MS
• 09 53 Scientist A looks at the microscope screen
• 10 05 Scientist A looking into microscope, reverse angle, MLS
• 10 16 Segment of the microscope is turning (and therefore turning the plane of sample), D
• 10 32 Scientist A and microscope, segment turning, MLS
• 10 57 Scientist A in front of the microscope, LS, scientist “working”
• 11 25 Resulting 3D section of a nucleoli of a yeast cell, magnification 19 thousand, 131 slices processed by computer into one video
• 11 59 Saccharomyces cerevisiae (yeast), 7800x magnification, 70 slices

Laboratory with confocal microscope

• 12 25 Scientist B in front of a confocal microscope, looking into ocular, MS
• 12 45 Scientist B in front of a confocal microscope, looking into ocular, MLS
• 12 53 Right hand touching controller, D
• 12 59 Scientist B in front of a confocal microscope, MLS, from behind
• 13 05 Face of a scientist B looking into ocular, D
• 13 25 Scientist B in front of a confocal microscope, looking into ocular, MLS
• 13 35 Finger pointing at a small display with data, data are changing, D
• 13 46 Scientist B in front of a confocal microscope, looking into ocular, MLS, reverse angle
• 12 52 Scientist B shifting from microscope to a computer display, MLS, pan to his hands and keyboard
• 14 06 Right hand manipulating with controllers D
• 14 14 Face of scientist B, D
• 14 45 Display with luminary cell from behind scientist B, MS
• 15 06 Left hand manipulating with controllers, D
• 15 35 Scientist B in front of a display, MLS, reverse angle
• 15 39 Luminary cells on display, green, micro
• 16 29 Luminary cells on display, dividing, accelerated, red, micro
• 16 44 Moving cells under microscope, accelerated, micro
• 17 05 Scientist C observing sample in a small dish, D
• 17 35 Scientist C placing a sample into a holder, (2x) MLS
• 18 06 Scientist C, reverse angle
• 18 13 Applying a solution onto the dish, MS
• 18 28 Placing a dish into microscope, MS
• 18 35 Placing a dish into microscope, D, reverse angle (6x)
• 19 48 Scientist C closing the microscope, MS
• 19 52 Sample in the microscope, D
• 20 02 Air bubbling through a cylinder, D
• 20 15 Air bubbling through a cylinder, reverse angle, focus change, D
• 20 27 Ocular, focus change, D
• 20 42 Dish with cells in microscope, D
• 20 57 Displays with different information, D
• 21 35 Scientists B and C in front of computer displays, pointing, pans at different details of the workplace, MLS

Preparing samples for the confocal microscope - microtome

• 22 36 Assistant taking solution of cells form an incubator, MS
• 22 52 Assistant taking solution of cells form an incubator – second take
• 23 08 Aspiration of solution by pipette, laminar box MS
• 23 17 Face of assistant, D
• 23 27 Aspiration of another solution from a small bottle – reverse angle
• 23 40 Releasing solution into a culture bottle, MS
• 23 59 Unwrapping a new glass piece, MS
• 24 12 Aspiration of a red solution, putting g into a culture bottle
• 24 33 Inspection of the solution, MS
• 24 50 Inspection of the solution, D
• 24 59 Face, D
• 25 05 Solution, D
• 25 09 Assistant preparing solution, MS
• 25 18 View from behind the glass, mixing the cell solution, MS – D
• 25 45 Face, D
• 25 50 Assistant sitting at the laminar box, MS
• 25 56 Assistant sitting at the laminar box, reverse angle, putting sample into the glass plate (this will be used at the confocal microscope)
• 26 15 Hand taking the glass vessel

Preparing the sample for the electron microscope

• 26 31 Scientist D at the microtome
• 26 37 Scientist taking sample of cells (embedded in a resin) from a dish and fixing into the microtome (ultra thin sectioning), MS
• 26 49 Fixing the sample into the device, D
• 27 17 Scientist looking into the ocular – checking the placing of the sample, MS
• 27 28 Changing the place of the holder, D
• 27 43 Putting holder into another place of the microtome, D
• 27 55 Taking one more part, where the water will be put, D
• 28 17 Scientist turning a controller, MS
• 28 32 Face of the scientist D, D
• 28 43 Water is pumped into the vessel, D – MS – D
• 29 08 Water table, D
• 29 28 Moving part is put closer to the tip with the sample, MS – D
• 29 50 Tip with the cell sample (embedded into the resin) is nearing the knife placed at the edge of the vessel, XCU
• 32 23 Tip with the cell sample is nearing the knife, MCU, zoom-in
• 36 46 Ultra thin slices are finally made, XCU
• 34 02 Scientist is taking the slice from the water surface using the grid (34 23)
• 34 40 The grid is put into the sample plate, D
• 35 22 Grid is put into the sample plate, second try, D
• 35 50 Grid is put into the sample plate, third try
• 35 05 Grid is put into the sample plate, fourth try

Exteriors

• 36 16 Exteriors, Building of the Institute of Cellular Biology and Pathology, VLS
• 36 05 Exteriors, Building of the Institute of Cellular Biology and Pathology, reverse angle
• 36 44 Exteriors, Building of the Institute of Cellular Biology and Pathology, reverse angle

D – Detail
MLS – Medium Long Shot (polocelek)
MS – Medium Shot
LS – Long Shot
VLS – Very Long Shot
XCU – Extreme Close Shot
MCU – Medium Close Up

Notes:

1. Directions of a cameraman and director (in Czech) can be heard in the background.
2. Images were shot at the Institute of Cellular Biology and Pathology, First Faculty of Medicine, Charles University, Prague [ http://lge.lf1.cuni.cz/emikr.html ]
3. Footage – courtesy Herafilm.cz (cameraman: Jaromir Herskovic)

C. Download two files: Training OK – 12.5MB and Training BAD – 9.3 MB). They represent two edited videos – view them, let them impress you and then answer the following questions:

1. Do you agree that the first cut is OK and the second BAD? Why?
2. What is missing? What is unnecessary?
3. From which cut can you get a better sense of the procedure?
4. Which cut makes an impression of chaos?
5. How do you assess the order of the takes (Detail, Medium Shot, Long Shot)
6. How do you judge rhythm of the takes (the length of takes, opportunity to realise what is going on)?
7. Are these cuts too long or too short? What criteria did you use?
8. Do some of the cuts feel strange or unnatural?

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