Widefield FLIM Bundle NEW

Fast cellular processes, moving particles, and rapid image acquisition have traditionally posed challenges for lifetime imaging using single-photon counting technologies. The Widefield FLIM Bundle addresses these limitations by enabling video-rate lifetime imaging within a widefield illumination setup. This bundle combines the high-resolution SPAD512² camera from Pi Imaging with PicoQuant's versatile pulsed laser portfolio and GPU-accelerated FLIM analysis through the NovaLITE software. Designed for compatibility with light-sheet and other widefield microscopy systems, it introduces FLIM capabilities via pulsed excitation and time-domain gated detection. Data analysis is streamlined with NovaLITE, optimized for the TIFF file format, ensuring efficient and accurate processing.

The bundle contains:

• One or multiple lasers for pulsed illumination at the desired wavelengths
• A 512x512 pixel SPAD array detector (SPAD512² high-speed single-photon camera from PI Imaging)
• Analysis software to perform fast FLIM, fitting, and phasor analysis
• Technical as well as application driven remote support

Read more about the contained components >

Principle of gated FLIM with an array detector

In confocal FLIM, the lifetime image is acquired sequentially by scanning over the pixels. The lifetime information in each pixel is acquired in parallel with TCSPC detection, building up the lifetime histogram. In contrast, in gated FLIM the entire image is acquired in parallel. The lifetime information, however, is acquired sequentially by shifting the gate relative to the fluorescence lifetime decay. The time-resolution is generated by discarding photons that fall outside the gate. Therefore, imaging speed, time-resolution, and even total detection efficiency depend on the acquisition parameters in a more complex way than in confocal FLIM.

Applications of Widefield Microscopy with FLIM >

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Applications of Widefield Microscopy with FLIM

Below, we highlight two key applications where our Widefield FLIM Bundle excels: Light-sheet FLIM and Widefield FLIM. Each showcases the bundle’s versatility in providing high-speed, high-precision imaging for advanced biological studies, from 2D widefield microscopy to 3D imaging of live samples using light-sheet techniques.

Light-sheet FLIM

In light-sheet microscopy (or selective plane illumination microscopy, SPIM) a plane of the sample is illuminated by a sheet of light, usually generated by a cylindrical lens or a beam scanning back and forth. The illuminated plane is then imaged onto a camera. Light-sheet microscopy is uniquely suited for studies on large living samples due to its speed, gentleness and 3D capabilities. The sensing or simultaneous multiplexing provided by lifetime imaging complements these advantages, making light-sheet FLIM a very potent combination.

In collaboration with researchers at the Aix-Marseille Université, we extended a light-sheet microscope to enable light-sheet FLIM. The microscope is a specialized single objective light-sheet, optimized for high throughput imaging of organoids. We combined this microscope with a fiber coupled VisUV488 for pulsed illumination and a SPAD512² sensor (high speed version) to perform FLIM on organoid samples.

The image shows how light-sheet FLIM can be used for simultaneous multiplexing. We imaged live organoids generated from mouse embryonic stem cells. Nuclei are expressing H2B-GFP and membranes are stained with Flipper-TR.

The video below shows 3D imaging of the organoids, multiplexed via lifetime. For this dataset, the light-sheet is generated from a gaussian beam scanning back and forth (approx. 70 µW average power at 488nm) and the acquisition time per Z-plane is 1.1 seconds. The scale bar is 50 µm.

For additional results including a comparison of different light-sheet modalities with confocal FLIM and 3D tension mapping of live organoids check the preprint on bioRxiv.

Widefield FLIM

In epifluorescence widefield microscopy, an area of the sample is illuminated by a collimated laser beam. The excited fluorescence is then imaged onto a camera. For widefield FLIM, we extended an epifluorescence microscope (Olympus IX73) with a fiber coupled high power 530 nm laser for illumination (LDH P-FA-530XL), and the high-speed version of the SPAD512² sensor.

The images show widefield FLIM and confocal FLIM of a commercial fixed cell sample (Gattaquant, scale bar 5µm). Mitochondria and tubulin are labelled with spectrally overlapping dyes but can be distinguished by lifetime. This lifetime contrast is visible even in a 40 ms acquisition on the widefield FLIM. The plot shows the broadening of the lifetime histogram with increasing imaging speed. The lifetime histograms from the widefield image with 300 ms acquisition time and the confocal image with 5 s acquisition time are both bimodal. However, the lifetime separation is larger in the confocal image. This is not an artifact of the gated acquisition, but rather a consequence of the lack of sectioning in epifluorescence microscopy, where the lifetimes of in-focus and out-of-focus contributions are mixed. The widefield FLIM images were recorded with an average laser power of ca. 10 mW at the laser output, corresponding to approximately 50 W/cm² in the field of view.

The high-speed of widefield FLIM is especially beneficial to avoid motion blur when imaging moving samples. The video shows large fluorescent beads free floating in water in real time (scalebar 10µm). The solution contains two different types of beads with similar spectral characteristics but differing lifetimes. In the video the two types of bead can clearly be distinguished by lifetime at a framerate of 23 frames per second.

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Components of the Widefield FLIM Bundle

Illumination Options

Widefield FLIM applications have very different demands regarding the illumination source, depending on illumination mode (e.g., Epi, TIRF, light-sheet, ...) and on your samples. In the applications tested so far, the necessary average laser power ranged from approximately 100 µW in a digitally scanned light-sheet system up to 25 mW in an epifluorescence widefield microscope. We will aid in selecting the right option for your application from the large portfolio of PicoQuant's pulsed laser sources.

For the most demanding applications, PicoQuant offers high power pulsed lasers with average power of over 35 mW (@20 MHz) at 488 nm, 530 nm, and 560 nm.

Standard Sources:

• LDH Series: Picosecond Laser Diode Heads
• Prima 3-Color Gain-Switched Picosecond Laser

High Power Sources:

• VisUV: Picosecond Lasers in UV, Green, Yellow, and Orange
• LDH-FA Series: Amplified Picosecond Pulsed Laser Diode Heads

Detector

The detector is the SPAD512² SPAD image sensor from Pi Imaging with 512x512 pixels. The sensor enables time-resolved detection with a nanosecond global exposure that can be shifted with picosecond precision relative to the laser pulse. It features microlenses for an increased fill factor and can be operated at overall count rates up to 15 Gcps.

*typically the bundle includes the standard version, high speed version may be available at longer lead-time

For more information on the sensor check the product page at Pi Imaging Technology.

Software

The analysis software NovaLITE reads in single gated decay measurements and performs average lifetime, phasor and fitting analysis. Users can visualize lifetime as histograms or in a phasor plot. The GPU-accelerated fitting allows fitting of single or multi-exponential decays and species separation based on fixed lifetimes within seconds. Results can be exported as TIFF for further processing. Note that NovaLITE is a pure analysis software, data acquisition is handled by the SPAD512² software, which users can integrate into their microscope via a remote interface. PicoQuant’s analysis software works with the universal TIFF file format. This is not the default format of the SPAD512² acquisition software, files need to be converted.

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