Dec 04, 2024
Photography of Algal Culture Vessels
- 1Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA;
- 2Quantitative Biosciences Institute, University of California, Berkeley, California 94720, USA
- Merchant Lab UC Berkeley
Protocol Citation: Dimitrios J. Camacho, Anne G. Glaesener, Sabeeha S. Merchant 2024. Photography of Algal Culture Vessels. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyw8jmvx9/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 02, 2024
Last Modified: December 04, 2024
Protocol Integer ID: 102695
Keywords: Photography, Algae, Chlamydomonas, Chromochloris, Auxenochlorella, Flask, Plate
Funders Acknowledgements:
National Institutes of Health (NIH): Nutritional Copper Signaling and Homeostasis Grant
Grant ID: GM 042143
National Institutes of Health (NIH): Molecular Basis of Cell Function T32 Training Grant
Grant ID: 5T32GM007232-44
US Department of Energy (DOE), Office of Biological and Environmental Research (BER): Systems Engineering of Auxenochlorella protothecoides: from Photosynthesis to Biofuels and Bioproducts Grant
Grant ID: DE-SC0023027
University of California, Berkeley, Chancellor’s Fellowship
Grant ID: N/A
Abstract
This protocol describes a method for photographing algal culture flasks, agar plates, and multi-well plates. The method describes the camera settings, imaging station set up, and image acquisition. Flasks are imaged from the side and plates are imaged from above. Manually defined settings and a color checker chart are used for increased consistency and reproducibility, allowing for cross comparisons of visual growth phenotypes across experiments.
Guidelines
Read the owner’s manual for the Canon EOS Rebel T3i or similar camera.
The camera settings described in this protocol can be optimized for your own purposes. The key is to keep the optimized settings constant throughout your experiments.
Materials
Camera:
Canon EOS Rebel T3i
Canon EFS 18 – 55 mm lens
Canon Battery pack LP-E8
Canon Battery charger LC-E8/LC-E8E
Canon Tripod
SD card
SD card reader
Imaging station:
Polaroid MP-4 Land Camera 44-01 Baseboard, vertical column, and lamps only (300 µmol photons m-2 s-1, 2700 K (warm white))
Calibrite ColorChecker Passport Photo 2 (CCPP2)
Calibrite ColorChecker Classic Mini
Xerox Vitality Colors Pastel Multipurpose Printer Paper 11x17", Color: Blue, Item # 156075, Office Depot
GE Healthcare Life Sciences GB 0056 Gel Blotting Paper 580 x 580 mm Cat no. 10426994
Software:
Adobe Lightroom Classic: https://helpx.adobe.com/lightroom-classic/get-started.html
Adobe Photoshop: https://www.adobe.com/products/photoshop/free-trial-download.html
Before start
Make sure that all flasks have labels that are above the liquid fill level. Orient the labels so that the manufacturer’s labels are to the rear. You will image the clear part of the flask facing forward.
The Lightroom Classic software uses a plug-in from the ColorChecker Camera Calibration software. The plug-in is automatically installed in Lightroom Classic if the ColorChecker Camera Calibration software has been installed.
Install Adobe Lightroom Classic: https://helpx.adobe.com/lightroom-classic/get-started.html
Camera settings for flask imaging
Camera settings for flask imaging
Turn the camera on and set the mode dial to "M" to enter the manual mode.
Top view of the Canon EOS Rebel T3i.
Press the Quick Control button and use the cross keys to navigate to the different settings.
Rear view of the Canon EOS Rebel T3i.
Use the following settings:
Shutter speed: 1/50
Aperture: F22
Picture style: Standard
White Balance: Tungsten light (Approx 3200K)
AF mode: One Shot
ISO speed: 400
Self-timer: 2 second delay
No flash
Quality: RAW + L 18M 5184 x 3456
Autocorrect image brightness and contrast: disabled
Note: These camera settings are for use with the polaroid baseboard and flood lamp set up. If you are using a different lighting set-up, you will need to adjust each setting.
Remove the dust cap from the lens and turn the focus switch to AF (auto focus) mode and switch on the stabilizer.
Side view of the Canon EOS Rebel T3i.
Imaging station set up for flask imaging
Imaging station set up for flask imaging
Make sure that the lights are turned off. The power switches are located on the front of the baseboard.
Lamp arm locking levers are located on each arm, but only the right side is labeled in the image.
Polaroid MP-4 Land Camera 44-01 baseboard, vertical column, vertical carriage, and lamps. Four 150 W, 120 V, Sylvania DWC flood lights are used for a combined output of approximately 300 µmol photons m-2 s-1 and a correlated color temperature of 2700 K (warm white). Lamp arm locking levers are located on each arm, but only the right side is labeled in the image.
Loosen the vertical carriage locking lever to unlock the vertical carriage. Use the height adjustment lever to raise the carriage above the lamps.
Loosen one lamp arm locking lever and carefully move the lamp arm inwards until the lamp arm stops rotating (see image in step 4). Repeat for the other lamp arm. Do one lamp arm at a time. Use the locking lever to lock the lamp arms in position.
To minimize shadows, position the lights close together and directed straight down. See image in step 4.
Next, position the inner lamps (left in the image below) on the inner notches along the horizontal bar. Position the outer lamps (right) on the outer limit of the horizontal bar.
Turn the lights on using the two power switches located in front of the polaroid land camera baseboard (see image in step 4).
Side view of lamps.
Install the camera to the tripod and press the live view shooting button to view the live feed on the LCD screen. Unfold and twist the LCD monitor so that the screen is visible.
Position the camera 30 cm from the baseboard. Use masking tape to mark the position of the tripod's feet on the floor.
Use the crank handle to raise and lower the center column, adjusting the height of the camera so that the surface of the baseboard is visible.
Tripod with parts labeled.
Tape a 580 x 580 mm Gel Blotting paper (Whatman paper) to the baseboard and lean the rest of the paper back to rest on the vertical column. The paper should be curved so that the background is blended (see image below).
Place a flask in the middle of the gel blotting paper (left to right) and 15 cm from the front edge of the baseboard and gel blotting paper.
Tape the Calibrite ColorChecker Classic Mini to the vertical column so that it is above the flasks. It is important that the ColorChecker is permanently affixed and does not move between sessions. Make sure that the ColorChecker is properly illuminated. See image below.
The color checker should be in frame of every image. See image in step 12.
Make sure that there are no shadows and that the flask(s) are illuminated evenly (see step 6 for position of lamps to avoid shadows).
a) Front view of flask imaging station. b) Side view of flask imaging station.
Adjust the height of the camera so that the flask is imaged directly from the side. See image in step 12. Use tape or a sharpie to mark the height of the center column on the twist lock of the tripod.
Flask image acquisition
Flask image acquisition
Verify all settings on the camera and that the lamps are positioned correctly.
Swirl the flask to mix the culture and ensure that cells do not settle.
You may need to let the culture sit for 5 - 10 s before imaging to remove all bubbles.
Place the culture flask on the imaging station. Minimize the amount of time that flasks are under the lights.
The flasks will get hot and condensation will form inside the flask if it is left illuminated too long.
Press the live view shooting button to view the live feed on the LCD screen.
Move the auto focus point (white box) to a flask using the cross keys.
Make sure that the flasks are completely within the white background.
Hold the shutter button down halfway and wait until the white box turns green to focus on the flask. When the camera is focused on the flask, press the shutter button down fully and let go of the camera. The image will be taken after 2 s.
To avoid motion artifacts, do not touch the camera or tripod while the shutter closes.
Unaltered raw photo of a flask captured using the specified camera settings.
To view the photos, press the playback button and use the left and right cross keys to move back and forward, respectively.
Camera settings for plate imaging
Camera settings for plate imaging
Use the following settings:
Shutter speed: 1/125
Aperture: F5.6
Picture style: Standard
White Balance: tungsten
AF mode: One Shot
ISO speed: 100
Self-timer: 2 second delay
No flash
Quality: RAW + L 18M 5184 × 3456
Autocorrect image brightness and contrast: disabled
Lens: Autofocus and stabilizer on
Set-up for plate imaging
Set-up for plate imaging
Make sure that the lamps are turned off.
Unlock one lamp arm locking lever and guide the lamp arm outwards.
The lamp will fall if not supported. Move each lamp arm separately.
Position the lamp arms so that they are perfectly vertical (90° in relation to baseboard).
Use the locking levers to lock the lamps in place. The lamps should be facing slightly inwards.
Unlock the vertical carriage by loosening the locking lever.
Lower the vertical carriage using the height adjustment lever so that it is just above the ColorChecker card that was taped to the column in step 8.
Install the camera to the mount and tighten the knob to secure the camera in place.
Place a blue sheet of paper on the baseboard.
Place your sample plate and the color checker passport on top of the blue paper.
Switch on the lamps. Limit the amount of time that the plate is illuminated by the lamps.
The plate will get hot and condensation will form inside the plate if the plate is left illuminated too long.
a) Front view of plate imaging set-up. b) Side view of plate imaging set-up.
Plate image acquisition
Plate image acquisition
Unfold and twist the LCD monitor on the camera so that it is visible (see image above of front view of the plate imaging set up).
Verify all camera settings.
Press the live view shooting button to view the live feed on the LCD screen.
Move the auto focus point (white box) to the plate using the cross keys.
Make sure that the Calibrite ColorChecker Passport Photo 2 is in frame.
Hold the shutter button down halfway and wait until the auto focus point turns green.
When the camera is focused on the flask, press the shutter button down fully and let go of the camera. The image will be taken after 2 s.
Unaltered raw photo of an agar plate containing Auxenochlorella protothecoides (UTEX 250).
Alternative imaging options
Alternative imaging options
If you do not have the components required in the first section, you may choose alternative methods listed in this section. A Calibrite ColorChecker or other color calibration charts with white, black, and gray scale references are required. Please note that while inexpensive color reference charts exist, they may not be recognized by the ColorChecker Camera Calibration plug-in of the Lightroom software. See the section titled, "Color correction for images taken with alternative methods" for more information regarding post processing of these images.
You may use any camera, including your cell phone, but verify that your camera meets your potential publisher's image resolution requirements.
Photograph your culture flasks or plates with the color chart in frame. Each photo must have the ColorChecker or other reference chart in it.
Image color correction
Image color correction
Remove the SD card from the camera and transfer the RAW and JPEG image files to a computer with Adobe Lightroom Classic and ColorChecker Camera Calibration software installed (See before you begin section for installation notes).
Import a photo of the Calibrite ColorChecker Passport Photo 2 into the Adobe Lightroom Classic Software.
Create a color profile by clicking on the "file" tab on the top left. Choose to "export with preset". Then choose the option "ColorChecker Camera Calibration" (See image below).
Enter a DNG Profile name in the dialogue box and click save.
The software will attempt to detect a color checker calibration chart in the photo and will automatically create a color profile with saved adjustments.
It will take a few seconds to a few minutes before the profile is generated. There will be no progress bar so you must be patient. Once generated, a dialogue box will appear stating that the profile was generated successfully.
Restart Adobe Lightroom Classic.
Import all other photos that have been taken using the same illumination set up and camera settings as those used when the image of the color checker was taken.
Click on the "Develop" tab on the top right of the screen.
Then select all photos on the bottom right of the screen that you wish to apply the color profile to.
Next, expand the "Basic" section of the toolbar to the right of the screen below the histogram.
Change the color profiles by clicking on the drop down menu where it reads "Profile: Adobe Color"
Select the name of the profile that you generated in step 24.
The color profile will be applied to all the photos selected.
Export the images by clicking on the "file" tab, then clicking "export".
Export the files to your hard drive and save the images as jpg, jpeg, or tiff files.
Import each photo in Adobe Illustrator or any other software that you use to generate publication quality figures.
Crop each photo so that only the portion of the flask containing liquid culture is visible.
Once all photos are cropped, adjust the sizes of all photos so that they are equal in length and width.
Arrange the photos and label them.
Keep a copy of the raw-unaltered photo files from the camera (RAW and JPEG files) in a separatorganized folder.
Color correction for images taken with alternative methods
Color correction for images taken with alternative methods
Download image files from the camera onto a computer with Adobe Photoshop installed.
Open each photo with a color checker or reference card in frame. You will have to manually apply a color correction filter to each individual photo using the steps below.
Click on the "image" tab on the top left of the screen, choose "adjustments", then "levels..."
A new window will pop up allowing you to make adjustments to the levels.
Click on the right most dropper to select a reference point for white. Click on the right most grid of the color checker in the image.
Next, click on the left most dropper to select a reference point for black. Click on the left most grid of the color checker grid in the image.
The software will correct the white balance, thus adjusting all other colors in the image as well.
Export each image as a jpeg, jpg, or tiff file.
Follow step 25 for cropping and sizing instructions.