The K2: Open-source simultaneous triple-color TIRF microscope for live-cell and single-molecule imaging

Niederauer, Christian; Seynen, Marco; Zomerdijk, Jan; Kamp, Marko; Ganzinger, Kristina A.

doi:10.1016/j.ohx.2023.e00404

Cited by 12 publications

(9 citation statements)

References 40 publications

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Section: Discussionmentioning

confidence: 99%

“…Many different modalities of homogenous TIRF microscopy were described based on continuous TIR ring illumination in the back focal plane [58, 59], galvometric laser scanning (spinning TIRF) [60-62] or multi-angular TIR illumination using digital mirror devices [63]. Combined with beam shaping devices as used for standard TIRFM [43, 64], these techniques would generate very homogenous flat-top TIR illumination. A recently published hybrid scanning and widefield method called Adaptable Scanning for Tunable Excitation Region (ASTER) reaches shadowless flat-top TIR illumination even for tunable big field of views [65].…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Correlative single-molecule and structured illumination microscopy of fast dynamics at the plasma membrane

Winkelmann,

Richter,

Eising

et al. 2023

Preprint

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Total internal reflection fluorescence (TIRF) microscopy offers powerful means to uncover the functional organization of proteins in the plasma membrane with very high spatial and temporal resolution. Traditional TIRF illumination, however, shows a Gaussian intensity profile, which is typically deteriorated by overlaying interference fringes hampering precise quantification of intensities – an important requisite for quantitative analyses in single-molecule localization microscopy (SMLM). Here, we combined flat-field illumination by using a standard πShaper with multi-angular TIR illumination by incorporating a spatial light modulator compatible with fast super-resolution structured illumination microscopy (SIM). This unique combination enabled quantitative multi-color SMLM with a highly homogenous illumination. By using a dual camera setup with optimized image splitting optics, we achieved versatile combination of SMLM and SIM with up to three channels. We deployed this setup for establishing robust detection of receptor stoichiometries based on single-molecule intensity analysis and single-molecule Förster resonance energy transfer (smFRET). Homogeneous illumination furthermore enabled long-term tracking and localization microscopy (TALM) of cell surface receptors identifying spatial heterogeneity of mobility and accessibility in the plasma membrane. By combination of TALM and SIM, spatially and molecularly heterogenous diffusion properties could be correlated with nanoscale cytoskeletal organization and dynamics.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Correlative single-molecule and structured illumination microscopy of fast dynamics at the plasma membrane

Winkelmann,

Richter,

Eising

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…The open microscopy hardware field is evolving rapidly 1 , 16 , 17 , 18 , 19 and the availability of low‐cost excitation sources based on LED or diode lasers is complemented by developments in low‐cost CMOS cameras. For fluorescence microscopy, including easySTORM , we are using two newly available cooled backside‐illuminated CMOS cameras based on circuitry originally developed for the amateur astronomy community and redesigned for the life sciences.…”

Section: Openframementioning

confidence: 99%

openFrame: A modular, sustainable, open microscopy platform with single‐shot, dual‐axis optical autofocus module providing high precision and long range of operation

Lightley,

Kumar,

Lim

et al. 2023

Journal of Microscopy

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Abstract“openFrame” is a modular, low‐cost, open‐hardware microscopy platform that can be configured or adapted to most light microscopy techniques and is easily upgradeable or expandable to multiple modalities. The ability to freely mix and interchange both open‐source and proprietary hardware components or software enables low‐cost, yet research‐grade instruments to be assembled and maintained. It also enables rapid prototyping of advanced or novel microscope systems. For long‐term time‐lapse image data acquisition, slide‐scanning or high content analysis, we have developed a novel optical autofocus incorporating orthogonal cylindrical optics to provide robust single‐shot closed‐loop focus lock, which we have demonstrated to accommodate defocus up to ±37 μm with <200 nm accuracy, and a 2‐step autofocus mode which we have shown can operate with defocus up to ±68 μm. We have used this to implement automated single molecule localization microscopy (SMLM) in a relatively low cost openFrame‐based instrument using multimode diode lasers for excitation and cooled CMOS cameras.This article is protected by copyright. All rights reserved

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“…1a). [21][22][23][24][25][26] In the marker-based approach, the fluorescence or scattering from bright particles is collected through a dedicated channel distinct from the imaging channel. 27,28 By monitoring these fiducial markers, their drift can be cor-rected.…”

Section: Introductionmentioning

confidence: 99%

Astigmatism-based focus stabilisation with universal objective lens compatibility, extended operating range and nanometre precision

Rahmani,

Cox,

Thamaravelil Abhimanue Achary

et al. 2024

Preprint

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Focus stabilisation is vital for long-term fluorescence imaging, particularly in the case of high-resolution imaging techniques. Current stabilisation solutions either rely on fiducial markers that can be perturbative, or on beam reflection monitoring that is limited to high-numerical aperture objective lenses, making multimodal and large-scale imaging challenging. We introduce a beam-based method that relies on astigmatism, which offers advantages in terms of precision and the range over which focus stabilisation is effective. This approach is shown to be compatible with a wide range of objective lenses (10x-100x), typically achieving <10 nm precision with >10 um operating range. Notably, our technique is largely unaffected by pointing stability errors, which in combination with implementation through a standalone Raspberry Pi architecture, offers a versatile focus stabilisation unit that can be added onto most existing microscope setups.

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The K2: Open-source simultaneous triple-color TIRF microscope for live-cell and single-molecule imaging

Cited by 12 publications

References 40 publications

Correlative single-molecule and structured illumination microscopy of fast dynamics at the plasma membrane

Correlative single-molecule and structured illumination microscopy of fast dynamics at the plasma membrane

openFrame: A modular, sustainable, open microscopy platform with single‐shot, dual‐axis optical autofocus module providing high precision and long range of operation

Astigmatism-based focus stabilisation with universal objective lens compatibility, extended operating range and nanometre precision

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