TABLE 1.

Example of PACVIER method utilizing 1% for the substratum and 0.1% for the bulk-medium interface^a

z-stack image no.c	Biomass area coverage (%)b
	Original stack (threshold = 43)	First iteration (threshold = 52)	Second iteration (threshold = 54)
1 (Substratum)	3.18	2.60	1.52
2	15.87	12.29	11.64
3	40.14	32.67	31.17
4	61.16	50.83	48.68
5	67.72	56.05	53.55
6	65.61	52.57	49.89
7	59.65	45.84	43.06
8	51.86	37.63	34.92
9	42.95	29.27	26.81
10	34.06	21.53	19.36
11	24.89	14.36	12.67
12	17.49	9.15	7.88
13	11.24	5.37	4.55
14	6.71	2.77	2.27
15	3.78	1.43	1.17
16	1.98	0.67	0.53
17	0.92	0.26	0.20
18	0.40	0.09
19	0.17	0.03
20	0.06
21	0.03
22	0.01
23	0.00
24	0.00

^aA threshold is calculated for the original z-stack by concatenating all of the images and performing the Otsu algorithm. Image removal begins at the substratum; images with a cellular area coverage value of <1% (or a value set by the user) are removed. Once an image with a cellular area coverage value of ≥1% is identified, it is set as the new substratum and the value of comparison switches to 0.1% (or a value set by the user) to identify the bulk-medium interface. Once an image with a cellular area coverage value of <0.1% is found, that image and all remaining images are removed from the stack (boldface values). A new Otsu threshold is calculated for the remaining images, and image removal is repeated. The process continues until the substratum image and bulk-medium interface image have a cellular area coverage value of greater than or equal to 1 and 0.1%, respectively.

^bTo calculate the cellular percent area coverage, each image is segmented using the calculated Otsu threshold. The number of pixels representing fluorescent signals is then divided by the total number of pixels in the image and multiplied by 100%.

^cThe z-step between images was 1 μm.