Core Biotechnology Services

Livecyte 2

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  • The system includes an image analysis packages which runs on the Livecyte Analysis computer also housed in MSB 385.

The Livecyte 2 is a label free imaging system using Quantitative Phase Imaging (QPI), but it also includes fluorescent LED channels for DAPI, CFP, GFP, YFP, TxRed and Cy5 to combine the QPI image with fluorescence images. This can be used for each time point, the start and/or end of the experiment or a subset of time points.

The system uses a QPI technique called ptychography to generate high contrast images from label free cells using very low intensity light, several magnitudes lower than used in traditional brightfield and fluorescence microscopy, significantly reducing phototoxicity. The system, therefore, allows long term live cell imaging, longer than 1 week if required. The results are high fidelity images which are artefact free and robustly quantitative, without the need for cell labelling.

The resulting images allow the quantification of various cellular features of individual cells. These include mitotic features, such as mitotic time and cumulative mitotic exit failure index, morphological features, such as cell dry mass, cell area, sphericity, thickness and length/width and kinetic features, such as speed, velocity and displacement. Collected data can be analysed and presented in changes in population metrics, for example, total dry mass, cell count, mitotic index and proliferation rate. The analysis software integrated in the system has highly robust analysis algorithms which allow for automatic identification and tracking of thousands of individual cells. Therefore, the system is capable of detecting subpopulations within the culture or single cells escaping a drug treatment. In this way subtle changes in cellular features, shape and movement can be quantitatively studied.

Objectives

  • PLN 10x/ NA=0.25 - Can be used with plastic bottom sample holders
  • SAPO 20x/ NA=0.75 - Needs glass bottom sample holders.

Sample holders

Currently, we can handle multi-well plates, 35 mm dishes and slides. There is a setup for a limited number of multi-well slides available. If other sample holders or slide types are required please contact Dr Kees Straatman.

Fluorescence

CoolLED pE 4000 LEDs available for fluorescence imaging:

  • DAPI: Ext LED 385 nm; Ext filter 350 - 400 nm; Em filter 410 - 480 nm.
  • CFP: Ext LED 435 nm; Ext filter 425 - 450 nm; Em filter 465 - 500 nm.
  • FITC: Ext LED 490 nm; Ext filter 470 -500 nm; Em filter 510 -540 nm.
  • YFP: Ext LED 500 nm; Ext filter 495 - 523 nm; Em filter 528 - 559 nm.
  • TxRed: Ext LED 550 nm; Ext filter 530 -580 nm; Em filter 600 - 650 nm.
  • Cy5: Ext LED 635 nm; Ext filter 603 - 650 nm; Em filter 670 - 715 nm.

Literature

  • Frame et al. (2017) J Cancer Metastasis Treat 3:302-14.
  • Kasprowicz et al. (2017) Int. J. Biochem. Cell Biol. 84:89–95.

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