time-lapse camera

Summary

Cells are cultured in culture chambers with suitable optical microscope characteristics. The microscope is fitted with a camera connected to a gas recorder, an additional device that provides the ideal temperature for cell culture. During recording, each exposure is triggered by a controller. The controller also controls the shutter in order to exclude unnecessary illumination of the cells.

Operation method

Program 27.4 Interval Camera

Principle

Cells are cultured in culture chambers with suitable optical microscope characteristics. The microscope is fitted with a camera connected to a gas recorder, an additional device that provides the ideal temperature for cell culture. During recording, each exposure is triggered by a controller. The controller also controls the shutter in order to exclude unnecessary illumination of the cells.

Materials and Instruments

Culture medium Trypsin and EDTA mixture
Coverslips Plastic Pierce Petri dishes Microscopes Unbreakable clear plastic cases Computer-controlled shutter and filter wheels Cameras Computers Imaging software IPlab

Move

1. Switch on all equipment, especially the heater. It is best to turn on the heaters first so that the clear plastic case (into which the microscope is enclosed) contains a controlled heating system. This controlled heating system is customized (RS Components, UK ) with a controller (2082793), temperature probe (2194674), heater element (224565) and low noise AC fan (583-325) to maintain a constant temperature inside.

2. Adherent cultured cells were digested with a mixture of trypsin and EDTA (0.25% trypsin (Difco) and 0.5 mmol/L EDTA) to make a cell suspension.

3. Coverslips (18 mm×18 mm , No 1.5 ( Merck ) in Picasso Petri dishes. After washing with concentrated sulfuric acid, the coverslips were repeatedly boiled in ultrapure water in Teflon coverslip containers (Eppendorf ) and finally sterilized in an autoclave.). Approximately 20,000 cells were inoculated on and the cells were left overnight at 37°C and under humidified conditions containing 3% _CO2.

4. Seal the coverslip over the Fibrex chambers. The chamber should be filled with culture medium (MEM, containing Hank's salt, 12 mmol/L sodium bicarbonate, 10% calf serum, and 4 mmol/L L-glutamine), leaving 1 small bubble. Seal the coverslips in situ with a hot wax mixture and leave 1 small bubble to ensure _CO2 equilibration by Hank's salt.

Phenanthrene chambers:

(a) 1 mm thick glass slide with a 15 mm diameter hole in the center. Cover the hole with a #3 coverslip and adhere the coverslip to the perforated slide with UV-treated glass adhesive (Southern Watch and Clock Supplies).

(b) Sterilize the chamber with 70 % ethanol and blow dry with clean air.

(c) Wax mixture: beeswax (Fisher ), soft yellow paraffin wax (Fisher ), and paraffin wax (melting point 46°C; Fisher ) in a 1 : 1 : 1 ratio.


5. Applying the appropriate microscope (Axiovert 135 TV ( Zeiss)) techniques, e.g., phase contrast and fluorescence with LD achromatic flat-field 32×/0.4Ph2 objective. High magnification objectives with shorter working distances need to be equipped with an orthostatic microscope in order to visualize adherent cells moving on top of the chamber.

6. Prepare the computer for image acquisition:

(a) Calibrate the image size with a microscope stage micrometer.

(b) Select the exposure time. Usually 0.1s is sufficient for phase contrast images, while weak fluorescence images require longer exposure times, 0.4s or more.

(c) Select the camera interval, number of images and file name. When analyzing the displacement of cells in slow motion, fibroblast-like cells will give satisfactory results in 5 min intervals. When observing the morphology of fast-changing cells, an interval of 1 min or even less will be required. Alternatively, displacement of fast-moving cells, such as neutrophils, will require intervals of 1 min or less.

(d) Follow the procedure below in order to alternate phase contrast images and fluorescence images:

(i)Create indexed file names for easy saving.

(ii) Insert the START marker.

(iii)Use the fluorescent filter wheel to open the arc lamp shutter.

(iv)Acquire the fluorescence image fully with appropriate exposure time.

(V) Close the arc lamp shutter using the phase contrast filter wheel.

(vi)Save the fluorescence image.

(VII) Open the halogen lamp shutter.

(viii)Acquire the phase contrast image fully with appropriate exposure time.

(ix)Close the halogen lamp shutter.

(x) Save the phase contrast image.

(xi)Pause interval recording.

(xii) Close all program windows.

(xiii) Eliminates the START mark unless a specified number of images have been recorded.

(xiv)Stop the program.

Phase contrast and fluorescence camera results recorded separately or in combination with IPLab software can be observed separately.

7. Store the image results on a CD burner.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/