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Definite focus

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Definite Focus is a high-precision focus control system developed by Zeiss. It automatically maintains the focal point of an imaging system, ensuring consistent focus during long-term imaging sessions.

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Lab products found in correlation

Axio observer z1, by Zeiss (15 mentions) Axio observer z1 inverted microscope, by Zeiss (4 mentions) Colibri 2, by Zeiss (2 mentions) Axiovision 4, by Zeiss (2 mentions) Plan apo objective na 0.8, by Hamamatsu Photonics (2 mentions) Imagem emccd camera, by Hamamatsu Photonics (2 mentions) Lsm 780, by Zeiss (2 mentions) Fluo 4 am, by Thermo Fisher Scientific (2 mentions) Lsm 880, by Zeiss (2 mentions) Lsm 710, by Zeiss (2 mentions) Lsm 780 nlo confocal microscope, by Zeiss (1 mentions) Fluorobrite dmem, by Thermo Fisher Scientific (1 mentions) Tempmodule s, by Zeiss (1 mentions) Ixon ultra 897, by Oxford Instruments (1 mentions) Zen 2.3 lite software, by Zeiss (1 mentions) Axiocam 506, by Zeiss (1 mentions) Co2 module s, by Pecon (1 mentions) Tempmodule s, by Pecon (1 mentions) Axio observer stand, by Zeiss (1 mentions) Cellasic onix2 microfluidic system, by Merck Group (1 mentions)

25 protocols using definite focus

1

Long-Term Live-Cell Imaging Microscopy

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Cells in the microfluidic device were imaged using a fully motorised Axio Observer Z1 inverted microscope (Zeiss), with constant focus maintained with focus stabilisation hardware (Definite focus, Zeiss). To minimise phototoxicity, we used LED light sources for both phase contrast and fluorescence images (Colibri 2, Zeiss) with the following parameters: 4.0 V – 70 ms for phase contrast and 15% of maximum intensity – 400 ms with 2 × 2 binning for the 560 nm LED. The temperature was maintained at 30°C with a controlled heating unit and an incubation chamber that held the entire microscope base, including the stage and the objectives. Images were acquired every 10 min using AxioVision 4 (Zeiss). All aspects of image acquisition were fully automated and controlled, including temperature, focus, stage position, and time-lapse imaging. Images were acquired for >120 h in standard experiments and up to 240 h in the experiment using telomere-elongated cells.
Xu Z., Fallet E., Paoletti C., Fehrmann S., Charvin G, & Teixeira M.T. (2015). Two routes to senescence revealed by real-time analysis of telomerase-negative single lineages. Nature Communications, 6, 7680.
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2

Live-cell imaging of nuclear factors

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Samples were imaged on a Zeiss AxioObserver Z1 inverted microscope using a Zeiss 20x plan apo objective (NA 0.8) using the appropriate filter sets and a Hamamatsu ImagEM EMCCD camera. Cells were maintained in a 37 degree incubation chamber at 5% CO2. Cells were imaged every 15 minutes. Focus was maintained using a combination of Zeiss Definite Focus and, using a custom script in MicroManager 2.0 beta (Edelstein et al., 2014 ), software autofocus adjustments every hour to compensate for slight movement of the membrane. For maximum accuracy, cells in this time-lapse were tracked manually in Fiji (Schindelin et al., 2012 (link)) (Figure 2B, n = 40; Figure 5B, FOXB2 transduced, n = 40; Figure 5B FOXB2 nontransduced controls, n = 30; Figure 5B CFP transduced, n = 32; Figure 5B CFP nontransduced controls, n = 44), and the tracks were analyzed with a custom python script that performed illumination profile correction. All mitotic events were captured because we were imaging nuclear transcription factors. Occasionally, a cell track could not be resolved confidently from the beginning to the end of the time-lapse, and any such tracks were truncated to cover only the high-confidence portion of the track.
Valcourt J.R., Huang R.E., Kundu S., Venkatasubramanian D., Kingston R.E, & Ramanathan S. (2021). Modulating mesendoderm competence during human germ layer differentiation. Cell reports, 37(6), 109990.
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3

Confocal and TIRF Microscopy Protocols

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Confocal microscopy was performed using a Zeiss LSM 780 NLO confocal microscope at 37°C, equipped with a 63 × 1.40 oil objective. Definite Focus (Carl Zeiss) was used to minimize focus shifts during time-lapse experiments. For excitation of sfGFP and mCherry 488 nm and 561 nm laser lines were used, respectively.
Time-lapse TIRF microscopy was performed at 37°C on an Olympus Biosystems CellˆTIRF system using an Olympus APON 100× oil TIRF objective (NA 1.49). To excite B-GECO1, sfGFP and mCherry, 405 nm, 488 nm and 561 nm solid state lasers were used, respectively. The laser alignment was performed individually for each region of interest. Image acquisition was operated by an xCELLence software package. The laser-based Z-drift compensator (ZDC) function was used to avoid focus shifts. Images were acquired every second for 4-5 min, with an exposure time of 100 ms.
Schifferer M., Yushchenko D.A., Stein F., Bolbat A, & Schultz C. (2017). A Ratiometric Sensor for Imaging Insulin Secretion in Single β Cells. Cell Chemical Biology, 24(4), 525-531.e4.
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4

Live-cell imaging of nuclear factors

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Samples were imaged on a Zeiss AxioObserver Z1 inverted microscope using a Zeiss 20x plan apo objective (NA 0.8) using the appropriate filter sets and a Hamamatsu ImagEM EMCCD camera. Cells were maintained in a 37 degree incubation chamber at 5% CO2. Cells were imaged every 15 minutes. Focus was maintained using a combination of Zeiss Definite Focus and, using a custom script in MicroManager 2.0 beta (Edelstein et al., 2014 ), software autofocus adjustments every hour to compensate for slight movement of the membrane. For maximum accuracy, cells in this time-lapse were tracked manually in Fiji (Schindelin et al., 2012 (link)) (Figure 2B, n = 40; Figure 5B, FOXB2 transduced, n = 40; Figure 5B FOXB2 nontransduced controls, n = 30; Figure 5B CFP transduced, n = 32; Figure 5B CFP nontransduced controls, n = 44), and the tracks were analyzed with a custom python script that performed illumination profile correction. All mitotic events were captured because we were imaging nuclear transcription factors. Occasionally, a cell track could not be resolved confidently from the beginning to the end of the time-lapse, and any such tracks were truncated to cover only the high-confidence portion of the track.
Valcourt J.R., Huang R.E., Kundu S., Venkatasubramanian D., Kingston R.E, & Ramanathan S. (2021). Modulating mesendoderm competence during human germ layer differentiation. Cell reports, 37(6), 109990.
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5

Myelin Phagocytosis Assay Protocol

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In vitro phagocytosis was analyzed by Fluoromyelin Green pre-labeling of myelin and fixation with 4% PFA. For fluorescence stainings, cell were permeabilized for 5 minutes with 0.1% TritonX-100, blocked for 30 minutes with 4% HS, 2% BSA in PBS and incubated with primary antibody (anti-Iba1, Wako Cat #019-19741, dilution 1:1000; anti-LAMP1, BD Pharmingen Cat #09671D, dilution 1:400) overnight. Cells were incubated with secondary antibodies in blocking solution for 1h followed by nuclear DAPI labeling. For analysis mean fluorescence intensity of Fluoromyelin-positive accumulations were quantified using CellProfiler 4.0, defining cells by DAPI and Iba1-positive staining. For phagocyte survival assay, live imaging was performed on an Axio Imager.Z1 (Zeiss) equipped with an AxioCam MRc3, x0.63 Camera Adaptor and the ZEN 2012 blue edition software using 20x objective (Plan-Apochromat x20/0.8) at 37°C and 5% CO2 (v/v). Cells were imaged over 16h with a frame rate of 5-8 minutes and Definite Focus (Zeiss). Analysis was performed with ImageJ 1.52p to determine survival rate.
Berghoff S.A., Spieth L., Sun T., Hosang L., Schlaphoff L., Depp C., Düking T., Winchenbach J., Neuber J., Ewers D., Scholz P., van der Meer F., Cantuti-Castelvetri L., Sasmita A.O., Meschkat M., Ruhwedel T., Möbius W., Sankowski R., Prinz M., Huitinga I., Sereda M.W., Odoardi F., Ischebeck T., Simons M., Stadelmann-Nessler C., Edgar J.M., Nave K.A, & Saher G. (2020). Microglia facilitate repair of demyelinated lesions via post-squalene sterol synthesis. Nature neuroscience, 24(1), 47-60.
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6

FRET Microscopy of Vinculin Tension

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Cells were transfected with VinTS. The day before the experiment, medium was changed to phenol-free (FluoroBrite DMEM, A18967; Gibco). Cells were wounded for 4 h, and Hepes was added before acquisition. Images were acquired with a Zeiss LSM780 with a 63× 1.4-NA oil objective and Definite Focus with Zen software at 5% CO2 and 37°C in phenol-free medium. The donor was excited at 458 nm, and the emission spectrum of the sensor was in 13 contiguous channels from 465 to 545 nm, to include both the donor and acceptor emission wavelengths. Images were analyzed through the Fiji plugin PixFret. After background correction, the FRET index was calculated on thresholded images to delineate the FAs. PixFret calculates the FRET index by measuring the ratio of intensity IFRET/(IDonor + IFRET) between the donor channel and the FRET channel (channels 3 and 6).
De Pascalis C., Pérez-González C., Seetharaman S., Boëda B., Vianay B., Burute M., Leduc C., Borghi N., Trepat X, & Etienne-Manneville S. (2018). Intermediate filaments control collective migration by restricting traction forces and sustaining cell–cell contacts. The Journal of Cell Biology, 217(9), 3031-3044.
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7

Live-Cell Imaging of CD63-Phluorin Dynamics

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Images were acquired with an Axio.ObserverZ1 motorized microscope (Zeiss) equipped with a CSU-X1 confocal scan head (Yokogawa) controlled by ZEN software (2.6; Zeiss). A C-ApoCHROMAT 63×/1.20 NA water immersion objective (Zeiss) was used with the following excitation and emission settings: CD63–phluorin, 488 nm excitation, 503–538 nm emission (50 ms exposure time). Images were collected with a QuantEM:512SC camera (Photometrics) and an image pixel size of 0.212×0.212 µm (xy). Images were collected every 5 min for 3 h and the focal plane was maintained with Definite Focus (Zeiss). Samples were maintained at 37°C with saturating humidity.
Gustafson C.M., Roffers-Agarwal J, & Gammill L.S. (2022). Chick cranial neural crest cells release extracellular vesicles that are critical for their migration. Journal of Cell Science, 135(12), jcs260272.
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8

Automated Long-term Live Cell Imaging

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Cells in the microfluidic device were imaged using a fully motorized Axio Observer Z1 inverted microscope (Zeiss), with constant focus maintained with focus stabilization hardware (Definite focus, Zeiss). To minimize phototoxicity, we used light-emitting diode (LED) light sources for both phase contrast and fluorescence images (Colibri 2, Zeiss) with the following parameters: 4.0 V–70 ms for phase contrast and 15% of maximum intensity −400 ms with 2 × 2 binning for the 560 nm LED. The temperature was maintained at 30 °C with a controlled heating unit and an incubation chamber that held the entire microscope base, including the stage and the objectives. Images were acquired every 10 min using AxioVision 4 (Zeiss). All aspects of image acquisition were fully automated and controlled, including temperature, focus, stage position and time-lapse imaging. Images were acquired for >120 h in standard experiments and up to 240 h in the experiment using cells with elongated telomeres.
Xu Z., Fallet E., Paoletti C., Fehrmann S., Charvin G, & Teixeira M.T. (2015). Two routes to senescence revealed by real-time analysis of telomerase-negative single lineages. Nature Communications, 6, 7680.
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9

Calcium Imaging of Midgut in Flies

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We used flies carrying esg-Gal4 10×UAS-mry:tdTomato 20×UAS-IVS-GCaMP6s for short-term calcium imaging. We loaded the female flies at 6 days after eclosion into the FlyVAB and we mounted them using HA 20 mg/ml. The posterior midgut was imaged using Zeiss Definite Focus. We acquired 6 µm thick Z-stack images (4 slices) at 256 × 256 pixels, with a pixel time of 2.06 μs and a total of 354 time points over 10 min.
Tang R., Qin P., Liu X., Wu S., Yao R., Cai G., Gao J., Wu Y, & Guo Z. (2021). Intravital imaging strategy FlyVAB reveals the dependence of Drosophila enteroblast differentiation on the local physiology. Communications Biology, 4, 1223.
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10

Automated Fluorescence Microscopy Imaging

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We imaged the plates automatically using an inverted fluorescence microscope (Axio Observer Z1; Zeiss, Oberkochen, Germany) equipped with a 20× objective (0.8 NA), a laser auto-focus system (Definite Focus; Zeiss, Oberkochen, Germany), and a scientific charge-coupled-device (CCD) camera (CoolSNAP HQ2; Photometrics, Tuscon, USA). We imaged four fluorescence channels: Ex: 365/Em: 465 nm (the “blue channel”; Zeiss filter set 49), Ex: 470/Em: 525 nm (the “green channel”; Zeiss filter set 38), Ex: 545/Em: 605 nm (the “red channel”; Zeiss filter set 43) and Ex: 628/Em: 692 nm (the “far-red channel”; Semrock Cy5-4040B). The exposure times were ~ 30–50 ms (the blue channel), ~ 1–3 s (the green channel), 500 ms (the red channel), and ~ 1–3 s (the far-red channel). Within each well, four images at different locations were acquired and saved in 16-bit TIFF format.
Lee J.Y., Miller J.A., Basu S., Kee T.Z, & Loo L.H. (2018). Building predictive in vitro pulmonary toxicity assays using high-throughput imaging and artificial intelligence. Archives of Toxicology, 92(6), 2055-2075.
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