# Free western blot quantification software (now that Image Studio Lite is gone)

> Image Studio Lite is discontinued. Quantify western blots free in ImageJ (Analyze › Gels), step by step, with Image Lab and IOCBIO Gel compared.

Published 2026-11-06 by LabCanvas team. https://www.labcanvas.com/blog/free-western-blot-quantification-software

![ImageJ 1.54f quantifying the synthetic western blot with Analyze › Gels: eight lane rectangles numbered on the blot (lane 1 with Select First Lane, lanes 2 to 8 with Select Next Lane) and the Gel Analyzer Options dialog with Invert peaks on for dark bands](https://www.labcanvas.com/blog/imagej/free-western-blot-quantification-software-imagej-gels.webp)

LI-COR's free **Image Studio Lite** is discontinued; its successor, **Empiria Studio**, is paid (with a 120-day trial). The free replacement most labs reach for is **ImageJ or Fiji**: draw a rectangle on each lane, **Analyze › Gels › Plot Lanes**, close each band's peak with a straight line and click it with the **Wand** to get its area, then divide by the loading control. **Bio-Rad Image Lab** (standard edition) and **IOCBIO Gel** are the other free options.

$$\text{Fold change} = \frac{\text{target} \div \text{loading control in the lane}}{\text{target} \div \text{loading control in the control lane}}$$

*Status checked in November 2026 on LI-COR's, Bio-Rad's and the IOCBIO Gel project's pages.*

## What happened to Image Studio Lite?

Image Studio Lite was LI-COR's free desktop program for quantifying western blots and gels, and many labs used it for blots from any imager. LI-COR discontinued it in favour of **Empiria Studio**; Wikipedia and the SciCrunch registry date the end of the free download to 2021. LI-COR's Image Studio Lite page now says the free software has been discontinued and offers a **120-day free trial of Empiria Studio**.

The same LI-COR page, in November 2026, also announces that **Image Studio 6.2** (the full acquisition and analysis software for LI-COR Odyssey imagers) is **free to download "for a limited time"**. If you have an Odyssey, that is worth a look; read the current terms on LI-COR's page first, since a limited-time offer can end.

Copies of Image Studio Lite still installed on lab computers keep working, but you cannot download it again, it gets no updates, and new colleagues cannot install it. That is why labs are looking for a replacement.

## ImageJ and Fiji: Analyze › Gels

The gel analyser is part of ImageJ 1.x, so it is the same in plain ImageJ and Fiji, and costs nothing. The short version (the full walkthrough is in [western blot quantification in ImageJ](https://www.labcanvas.com/blog/western-blot-quantification-imagej)):

1. Open the original image (a 16-bit TIFF if your imager saves one). Check **Analyze › Gels › Gel Analyzer Options…**: **Invert peaks** on for dark bands on a light background.
2. Draw a rectangle over lane 1 and press **1** (**Select First Lane**). Move the same rectangle to each other lane and press **2** (**Select Next Lane**).
3. Press **3** (**Plot Lanes**): one profile per lane, each band a peak.
4. With the straight-line tool, close each peak at its foot; this line is the background.
5. Click inside each closed peak with the **Wand**: its area goes to the Results table. **Label Peaks** adds the percentages.
6. In a spreadsheet, divide each target band by the loading control in the same lane, then by the control lane's ratio.

![ImageJ with eight lane rectangles on a western blot and the Gel Analyzer Options dialog](https://www.labcanvas.com/blog/imagej/free-western-blot-quantification-software-imagej-gels.webp "ImageJ 1.54f on the synthetic western blot: lane 1 selected with Select First Lane 1, lanes 2 to 8 with Select Next Lane 2, numbered on the blot. In Gel Analyzer Options, Invert peaks is on for dark bands 3.")

![ImageJ Plot Lanes window with one profile per blot lane](https://www.labcanvas.com/blog/imagej/ij-gels.webp "ImageJ: Analyze › Gels › Plot Lanes on the western blot sample, one profile per lane; each band is a peak to close with a straight line and click with the wand.")

### Worked example: the synthetic blot in ImageJ

The sample blot is synthetic, with known amounts: eight lanes, a target band that rises across them, and a loading control with small lane-to-lane differences, as on a real blot. The true fold change of lane 8, from the amounts used to make the image, is 8.935.

For a result that can be repeated exactly, we measured it in ImageJ 1.54f with a short macro that does what the Gels steps do: the profile down each lane (the average across a 48-pixel-wide lane), a straight baseline from the background on one side of each band to the other, and the area between them. Lane 1 is the control.

![ImageJ table of target and loading-control areas per lane with the ratio and fold change](https://www.labcanvas.com/blog/imagej/free-western-blot-quantification-software-imagej-fold-change.webp "ImageJ 1.54f on the synthetic blot: target and loading-control band areas above a straight baseline in each lane, their ratio, and the fold change against lane 1 1. Lane 8 comes out at 8.736-fold 2.")

| Lane | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
|---|---|---|---|---|---|---|---|---|
| Target ÷ loading control | 0.222 | 0.460 | 0.749 | 1.142 | 1.616 | 1.629 | 1.655 | 1.938 |
| Fold change (ImageJ) | 1.000 | 2.071 | 3.375 | 5.146 | 7.282 | 7.339 | 7.460 | 8.736 |

Lane 8 at 8.736-fold is within 2.5% of the true 8.935. The small shortfall comes from where the baseline sits and how much of each band's tails falls inside the window, not from the program. Place the baseline the same way in every lane and every blot.

## The free options compared

| Software | Price | Runs on | Lanes and bands | Background | Normalisation and fold change | Best for |
|---|---|---|---|---|---|---|
| **ImageJ / Fiji** (Analyze › Gels) | Free, open source | Windows, macOS, Linux | Manual: you draw each lane, close each peak by hand | A straight line you draw under each peak | In a spreadsheet | Any image, full control, a method every reviewer knows |
| **Bio-Rad Image Lab** (standard edition) | Free download | Windows, macOS | Automatic lane and band detection | Built in | Built in, including stain-free total-protein normalisation | Blots from Bio-Rad ChemiDoc and Gel Doc imagers |
| **IOCBIO Gel** 1.1.1 | Free, open source (GPLv3) | Windows (installer), any system through Python (pip) | Lanes you set, integration limits you set | Background subtraction, recorded | Measurements exported for your own normalisation | Reproducibility: every step and the original image stored in a database |
| **Empiria Studio** (LI-COR) | Paid; 120-day trial | Desktop | Automatic | Built in | Built in | Labs on LI-COR imagers that want the full workflow |
| **Image Studio 6.2** (LI-COR) | Free for a limited time (Nov 2026) | Desktop | Automatic | Built in | Built in | Owners of LI-COR Odyssey imagers |

What none of them can do is rescue a saturated or over-exposed blot. Quantify the original file from the imager, check that no band is clipped, and make sure the bands sit in the linear range of your detection (Pillai-Kastoori et al. 2020).

## Bio-Rad Image Lab

Image Lab is Bio-Rad's software for its ChemiDoc and Gel Doc imagers, and Bio-Rad offers the **standard edition as a free download** for analysis on any computer (the Security Edition, for 21 CFR Part 11 work, is licensed). It detects lanes and bands automatically, subtracts background, reads molecular weights from a standard lane, and normalises to a housekeeping protein or to total protein on stain-free gels, with a report at the end.

It is at its best with Bio-Rad's own image files, which carry the exposure and saturation information. If your blots come from another imager, try a few in the free version before you commit.

## IOCBIO Gel

IOCBIO Gel is an open-source program from the Laboratory of Systems Biology at Tallinn University of Technology (Kütt et al. 2023). Version 1.1.1 was released in October 2025; it comes as a Windows executable and through the Python Package Index for other systems, under the GPLv3 licence.

Its point is reproducibility: it stores the original image and every operation on it (cropping, background subtraction, lane selection, integration limits) in a database, local for one person or shared for a team, so you can go back and correct any step months later. It was written for western blots but suits any gel-like image, such as isoelectric focusing. A step-by-step protocol was published in Bio-protocol in 2024 (Jaska et al. 2024).

## Which one to choose

- **You want the method every reviewer knows, on any image:** ImageJ or Fiji, Analyze › Gels.
- **Your blots come from a Bio-Rad imager:** Image Lab, standard edition.
- **You need an audit trail of every step:** IOCBIO Gel.
- **You have a LI-COR Odyssey:** Image Studio 6.2 while it is free, or Empiria Studio.

## Pitfalls, whichever software you use

- **Saturated bands.** A band whose brightest pixels hit the detector's limit has a flat top and is underestimated. Re-image with a shorter exposure; no software fixes it.
- **The linear range.** Check it once per antibody with a dilution series of a pooled sample (Pillai-Kastoori et al. 2020). Abundant loading controls such as GAPDH and β-actin reach the top of their range at much lower loads than most targets, so check them too.
- **JPEGs and screenshots.** Compression and contrast adjustments change band intensities. Quantify the original file.
- **Different background methods.** Rolling-ball, straight-line and no background give different numbers. Choose one and use it for every lane and every blot.
- **One blot.** Report at least three independent blots and test them statistically.

## Reporting it

A methods sentence to copy, with the parts in brackets filled in:

> Western blots were imaged on [imager] and quantified by densitometry in [ImageJ 1.54 (Analyze › Gels) / Image Lab 6 / IOCBIO Gel 1.1] on the unadjusted [16-bit TIFF] files, with a straight-line background under each band. No band was saturated. Target bands were normalised to [total protein / β-actin] in the same lane and expressed as fold change relative to [the control lane]; data are mean ± SD of [n = 3] independent blots.

Show the result as one point per blot with the mean ± SD, beside a representative blot with the molecular-weight markers labelled. The full ImageJ method is in [western blot quantification in ImageJ](https://www.labcanvas.com/blog/western-blot-quantification-imagej). Cite ImageJ as Schneider et al. (2012).

## An easier way: quantify the blot in LabCanvas

If drawing lanes and closing peaks by hand is the slow part, the **Blots** tab of [LabCanvas Image Analysis](https://www.labcanvas.com/analysis.html) does the same densitometry in the browser, with nothing to install.

1. Open your TIFF, or click the western blot sample on the start screen, and go to the **Blots** tab.
2. **Lanes**: **Find lanes** (or **Add lane**, or **Use selected** rectangles); mark a **Molecular-weight ladder** lane if you want band sizes.
3. **Bands**: **Find bands**, then drag and name the rows and mark the loading control; **Align lanes** corrects smiling lanes.
4. **Quantify**: choose a **Background** (**Straight line**, **Rolling** or **None**), **Normalise to** the loading control or a total-protein stain image, and set the **Reference lane**. The table gives area, normalised value and fold change.

![Western blot densitometry in LabCanvas Image Analysis](https://www.labcanvas.com/analysis-examples/blot.webp "Western blot densitometry: 8 lanes; the target band normalised to the loading control rises from 1 lane 1 to 8.885-fold lane 8.")

On the same synthetic blot, LabCanvas gives lane 2 at 2.103-fold and lane 8 at **8.885-fold**, against 2.071 and 8.736 in ImageJ and a true 8.935 for lane 8: LabCanvas within 1%, the ImageJ baseline method within 2.5%. The two differ by where each baseline sits and how wide each band window is.

Why it is a good choice for blots:

- **Nothing to install**: any computer or tablet with a modern browser, so new colleagues are not stuck without Image Studio Lite.
- **No spreadsheet step**: normalisation, the reference lane and fold change are in the table, and update as you move a band.
- **An image check first**: clipped (saturated) bands are flagged before you measure.
- **Straight into Graphs**: **Send to Graphs** takes the fold changes of replicate blots to the statistics, and the **Methods** sentence under the table is ready to copy. Quantifying, **Copy** and **Send to Graphs** are free with a LabCanvas account; downloading CSV files is part of LabCanvas Pro.
- **Checked**: every measurement is validated in [more than 4,000 automated tests](https://www.labcanvas.com/image-analysis-validation).

[Open Image Analysis](https://www.labcanvas.com/analysis.html). ImageJ and Fiji are independent open-source projects; LabCanvas is not affiliated with them, or with LI-COR or Bio-Rad.

## References

- Jaska L, Birkedal R, Laasmaa M, Vendelin M (2024). Simple analysis of gel images with IOCBIO Gel software. *Bio-protocol* 14, e5053. [doi:10.21769/BioProtoc.5053](https://doi.org/10.21769/BioProtoc.5053)
- Kütt J, Margus G, Kask L, Rätsepso T, Soodla K, Bernasconi R, Birkedal R, Järv P, Laasmaa M, Vendelin M (2023). Simple analysis of gel images with IOCBIO Gel. *BMC Biology* 21, 225. [doi:10.1186/s12915-023-01734-8](https://doi.org/10.1186/s12915-023-01734-8)
- Pillai-Kastoori L, Schutz-Geschwender AR, Harford JA (2020). A systematic approach to quantitative Western blot analysis. *Analytical Biochemistry* 593, 113608. [doi:10.1016/j.ab.2020.113608](https://doi.org/10.1016/j.ab.2020.113608)
- Schneider CA, Rasband WS, Eliceiri KW (2012). NIH Image to ImageJ: 25 years of image analysis. *Nature Methods* 9, 671–675. [doi:10.1038/nmeth.2089](https://doi.org/10.1038/nmeth.2089)

## Frequently asked questions

### Is Image Studio Lite still available?

No. LI-COR discontinued the free Image Studio Lite (Wikipedia dates this to 2021) in favour of Empiria Studio, which is paid and offers a 120-day free trial. In November 2026 LI-COR's Image Studio Lite page also offers Image Studio 6.2 as a free download "for a limited time"; check the page for the current terms.

### What is the best free alternative to Image Studio Lite?

For most labs: ImageJ or Fiji (Analyze › Gels) if you are happy with a manual, step-by-step method; Bio-Rad Image Lab (standard edition, free) if your blots come from a Bio-Rad imager; and IOCBIO Gel if you want every step recorded for reproducibility.

### Can I quantify western blots in ImageJ for free?

Yes. ImageJ and Fiji are free, and Analyze › Gels measures band densities: select the lanes, plot their profiles, close each peak with a straight line and click inside it with the Wand. You then normalise to a loading control and compute fold changes in a spreadsheet.

### Is Bio-Rad Image Lab free?

The standard edition of Image Lab is a free download from Bio-Rad and can analyse images on a computer without an imager. The Security Edition, for 21 CFR Part 11 compliance, needs a paid licence.

### Which image file should I quantify?

The original file from the imager, usually a 16-bit TIFF or the instrument's own format, never a JPEG, a screenshot or an image with the contrast adjusted. Check that no band is saturated, and stay in the linear range of the detection.

### Can I quantify a western blot without installing anything?

Yes: the Blots tab of LabCanvas Image Analysis finds lanes and bands, subtracts the background and gives fold change in the browser, free with an account; see the last section of this article.

## Related links

- [Western blot quantification in ImageJ, step by step](https://www.labcanvas.com/blog/western-blot-quantification-imagej): The full Analyze › Gels walkthrough
- [Western blot molecular weight calculator](https://www.labcanvas.com/tools/western-blot-mw-calculator): Band sizes from the ladder
- [Best ImageJ alternatives](https://www.labcanvas.com/blog/imagej-alternatives): Free, online and desktop
- [Microscopy image integrity](https://www.labcanvas.com/blog/microscopy-image-integrity): What you may and may not adjust
- [Image Analysis: the complete guide](https://www.labcanvas.com/blog/image-analysis-complete-guide): The Blots tab and every other tool